Patent Publication Number: US-9423396-B2

Title: Bodipy structure fluorescence probes for diverse biological applications

Description:
RELATED APPLICATION(S) 
     This application is the U.S. National Stage of International Application No. PCT/SG2012/000483, filed on Dec. 20, 2012, which designates the U.S., published in English, and claims the benefit of U.S. Provisional Application No. 61/579,461, filed on Dec. 22, 2011. The entire teachings of the above application(s) are incorporated herein by reference. 
    
    
     INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE 
     This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith: 
     a) File name: 44591040002_FINALSEQUENCELISTING.txt; created Jun. 18, 2014, 2 KB in size. 
     BACKGROUND OF THE INVENTION 
     Fluorescent probe molecules have been widely used in bioimaging and medicinal applications for several decades due to their high sensitivity and easy visibility. During the past few decades, a number of small molecule fluorescent chemosensors have been developed for use in biological analyses, which typically are elaborately designed to selectively detect a target substance or phenomenon. 
     Most fluorescent chemosensors employ increase or decrease in their emission intensity as a sensing signal in response to the surrounding medium or through specific molecular recognition events. Due to their simplicity and high sensitivity, fluorescent sensors have been widely utilized as popular tools in chemical, biological, and medical applications. 
     The conventional bioprobe design is based upon a hypothesis-driven approach. The conventional bioprobe usually consists of three parts: a target recognition motif, a linker and a fluorophore. The target recognition motif is designed based on the identity and structure of the target, and then is paired with any number of fluorophores to generate a bioprobe that will efficiently and selectively allow visualization or staining of a target. The basic assumption of this hypothesis-driven approach is that the scientist knows the target in advance, and then designs the recognition motif tailored specifically for the target. Therefore, the scope of bioprobe development is intrinsically limited by the available knowledge for the target. An alternative to this process is combinatorial dye library synthesis, which enables a scientist to arrive at a library of diversity directed sensors. 
     There remains a need to develop a library of fluorescent sensors that may be used in crucial biomedical applications, such as the selective imaging of pancreatic islets or microglia cells. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to novel BODIPY compounds represented by structural Formula (I) or pharmaceutically acceptable salts thereof: 
                         
wherein:
     R 1  is hydrogen or —COR 3 ;   R 2  is (C 6 -C 16 )aryl, (C 3 -C 10 )heteroaryl, (C 1 -C 6 )alkyl, (C 1 -C 6 )cycloalkyl, (C 2 -C 6 )alkenyl, or C≡CH;   R 2  is optionally substituted with 1-5 substituents independently selected from (C 1 -C 6 )alkyl, halogen, amino, cyano, —COOH, halo(C 1 -C 6 )alkyl, hydroxy(C 0 -C 6 )alkyl, (C 6 -C 10 )aryl, (C 3 -C 10 )heteroaryl, halo(C 6 -C 10 )aryl, hydroxy(C 6 -C 10 )aryl, (C 1 -C 6 )alkoxy, halo(C 1 -C 6 )alkoxy, (C 6 -C 16 )aryloxy, (C 3 -C 8 )cycloalkyl, halo(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, (C 1 -C 6 )alkoxy(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, nitro, (C 0 -C 6 )alkyl(C 6 -C 10 )aryl(C 0 -C 6 )alkoxy, (C 5 -C 10 )heterocycle, —OCHF 2 , —OCF 3 , —SCF 3 , —OBn, cyano(C 1 -C 6 )alkylene, (C 1 -C 6 )alkoxyamino, (C 6 -C 10 )aryl(C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 1 -C 6 )alkoxy, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 6 -C 10 )aryl, —N((C 0 -C 6 )alkyl)((C 1 -C 6 )alkyl), —N((C 1 -C 6 )alkyleneOH)((C 1 -C 6 )alkyleneOH), —N((C 0 -C 6 )alkyl)((C 1 -C 6 )alkyleneOH), —N((C 1 -C 6 )alkyleneOCO(C 1 -C 6 )alkyl)((C 1 -C 6 )alkyleneOCO(C 1 -C 6 )alkyl), —NCO(C 1 -C 6 )alkyl, —NPh 2 , —OPh(halogen) 0-3 , —OPhO(C 1 -C 6 )alkyl, —OPhO(C 1 -C 6 )alkyl, —OCO(C 1 -C 6 )alkyl, —OCO(C 1 -C 6 )alkoxy, —O(C 1 -C 6 )alkyl(C 6 -C 10 )aryl, —O(C 2 -C 6 )alkenyl, —O(C 2 -C 6 )alkyleneN(CH 3 ) 2 , (C 0 -C 6 )alkylCOO(C 1 -C 6 )alkyl, —B(OH) 2  or —S(C 1 -C 6 )alkyl; and wherein any of the substituents selected from (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryl, (C 6 -C 16 )aryloxy or (C 5 -C 10 )heteroaryl is further optionally substituted with 1-4 substituents selected from halogen, (C 1 -C 6 )alkyl, halo(C 1 -C 6 )alkyl, amino, nitro, cyano, hydroxy(C 0 -C 6 )alkyl, (C 1 -C 6 )alkoxy, —COO(C 0 -C 6 )alkyl, or —CHO; and   R 3  is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 6 -C 10 )aryl or (C 5 -C 10 )heteroaryl, wherein R 3  is optionally substituted with 1-4 substituents independently selected from halogen, amino, cyano or hydroxyl.   

     In one embodiment of the invention, R 2  is (C 6 -C 10 )aryl, and in another embodiment, R 2  is phenyl, optionally substituted with 1-4 substituents selected from —C(CH 3 ) 3  and —O—(C 1 -C 3 )alkyl. In a preferred embodiment of the invention, the compound has the structure of formula (II): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, the compound has the structure of formula (III): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, the compound has the structure of formula (IV): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, the compound has the structure of formula (V): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, the compound has the structure of formula (VI): 
     
       
         
         
             
             
         
       
     
     The invention is further directed to pharmaceutical compositions comprising a compound of any of formulae (I) through (VI) and one or more pharmaceutically acceptable carriers and/or one or more pharmaceutically acceptable diluents. 
     The present invention further provides methods for the solid phase synthesis of compounds of structural formulae (I) through (VI), comprising:
         (a) reacting a compound of structural Formula (VII):       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 with a base and an aldehyde, such that the activated C 3 -methyl group of Formula (VII) is modified in a solid-phase Knoevenagel-type, reaction to produce a compound of Formula VIII: 
               
             
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             b) removing the solid support resin from the compound of structural Formula (VIII) produced in step (a) to generate a BODIPY structure of Formula (IX): 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
              and 
             c) optionally reacting the compound of structural Formula (IX) with an acid chloride of the formula R 1 (CO)Cl to generate a compound having the structure of one of formulae (I) through (VI). 
           
         
       
    
     The present invention further describes methods for detecting beta cells using image based screening. The methods comprise (a) contacting a sample comprising cells with a compound of structural Formula (I) or pharmaceutically acceptable salts thereof; (b) incubating the sample and the compound of step (a) together for a period of time sufficient to stain the cells; and (c) analyzing the incubated stained cells by spectroscopy to detect a fluorescence signal, wherein the presence of a fluorescence signal is indicative of the presence of the beta cells. In a preferred embodiment of the invention, the method utilizes a compound of formula (II) in the detection of beta cells. In another preferred embodiment of the invention, the method utilizes a compound of formula (III) in the detection of beta cells. 
     The present invention further describes methods for detecting microglia cells comprising (a) contacting a sample comprising cells with a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof; (b) incubating the sample and compound of step a) together for a period of time sufficient to stain the cells; and (c) analyzing the incubated stained cells by spectroscopy to detect a fluorescence signal, wherein the presence of a fluorescence signal is indicative of the presence of microglia cells. In a preferred embodiment of the invention, the method utilizes a compound of formula (VI) in the detection of microglia cells. The invention further provides for methods of detecting microglia cells, in which activated microglia cells are distinguished from resting microglia cells by intensity of their fluorescence signals. 
     The present invention is further directed to methods for fluorescence imaging of pancreatic islet cells to determine health status, comprising (a) contacting the pancreatic islet cells with a compound of structural Formula (I) or pharmaceutically acceptable salts thereof; (b) incubating the cells and compound of step (a) together for a period of time sufficient for to stain the cells; (c) analyzing the incubated stained pancreatic islet cells by spectroscopy to detect a fluorescence signal; and (d) comparing the signal from step (c) to a fluorescence signal from reference sample of healthy pancreatic islet cells, to determine health status of the pancreatic islet cells. In a preferred embodiment of the invention, a compound having the structure of formula (III) is utilized in the fluorescence imaging of pancreatic islet cells. 
     The present invention provides that the fluorescence signal that is analyzed in the detection of beta cells, microglia cells, or pancreatic islet cell imaging is measured by fluorometer, UV/VIS spectrometer, Gemini XS fluorescence plate reader, a flow cytometry or a confocal microscope. Further, each of these methods are applied in vitro or alternately, in vivo. 
     The novel BODIPY dyes described herein are chemical compounds with fluorescence emission properties that enable staining of specific cell types and organs. Candidate compounds have high potential for the development of an in vivo islet imaging probe for pancreatic transplanted patient and microglia related inflammation imaging probe. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings. 
         FIGS. 1 a -1 c    show images of cellular detection utilizing the beta cell probe BDNCA325.  FIG. 1 a    shows the chemical structure of BDNCA325.  FIG. 1 b    shows the selective detection of a Beta TC-6 cell by BDNCA325 in contrast to Hoechst 33342 for nuclei staining. Specifically, the upper panel shows fluorescent (FL) images for Texas Red filter and the lower panel shows fluorescent (FL) images for DAPI filter.  FIG. 1 c    shows the visualization of a pancreas section by BDNCA325 in vitro tissue staining. The same images for BDNCA325 were acquired before immunostaining with insulin and merged for identification of islets. Scale bar, 100 μm. 
         FIGS. 2 a -2 c    show images of visualization of pancreatic islets by PiY in vivo staining.  FIG. 2 a    shows islets of pancreas cryo-tissues after administration of PiY observed under low magnification fluorescent microscopy. Scale bar, 200 μm. The fluorescent image was taken by TRITC filter and the areas of islets were marked by red dots in bright field (B.F) and fluorescence panel.  FIG. 2 b    shows the chemical structure of PiY.  FIG. 2 c    shows PiY-stained tissues that were immunostained using insulin and then co-localized for confirmation. Scale bar, 100 um. 
         FIGS. 3 a -3 d    show the development of PiY using streptozotocin-induced diabetic mouse model.  FIG. 3 a    shows comparative fluorescent images to exhibit differences between undamaged and damaged islets with PiY. These were used for analysis and the intensity size was examined using ROI software program. Scale bar, 100 μm.  FIG. 3 b    shows that islets obtained from STZ mice have few cells. This is demonstrated by decreasing fluorescent intensity of PiY and confirmed with insulin antibody. Each fluorescent image was merged for identification of islets.  FIG. 3 d    shows the diabetic animal model for type 1, generated by STZ administration. 
         FIG. 4  shows live primary microglia enriched cultures and neuron enriched neural cultures, stained with 500 nM of BDNCA1 H5 and imaged using a Texas red filter. This was followed by fixation and immunostaining for the microglia cell surface marker, Iba (green) and the corresponding neuron (β-tubulin), astrocyte (GFAP) and oligodendrocyte (O4) markers. 
         FIG. 5  shows primary microglia from mixed primary neural cell cultures derived from CX3CR1-GFP transgenic mice, stained with 500 nM of BDNCA1 H5 respectively (red). Hoescht staining was used for the labeling of cellular nuclei (Blue). 
         FIGS. 6A  and B show cytokine expression of compound-treated BV2 microglia. BV2 cells were pretreated for 1 h with 500 nM of compound before addition of 100 ng/ml of LPS in fresh media. After 6 hours, total RNA was isolated from the cells and cytokine expression levels of IL-6 and TNF-α were measured by RT-PCR. 
         FIGS. 7A and 7B  show results of an MTS assay used to assess cell viability after compound staining in cytotoxicity testing.  FIG. 7A  shows that BDNCA1 H5 has minimal cytotoxicity when used at concentrations of 1 uM and above.  FIG. 7B  is a measure of cell proliferation in the presence of compound staining. BDNCA1 H5 does not appear to affect cell proliferation as reflected by the robust increase in cell numbers over 48 hours. 
         FIG. 8A  shows nitric oxide production of compound and LPS/IFN-γ treated microglia. BV2 cells were pretreated for 1 h with 500 nM of compound before addition of 100 ng/nl of LPS or 20 U/ml (20 ng/ml) of IFN-γ in fresh media. Cells were further incubated for 24 h. Nitric oxide production was subsequently measured by the Griess assay and concentrations determined by comparison to a standard curve. In  FIG. 8B , compounds were accessed for potential activating effects on the microglia by measuring nitric oxide production. BV2 cells were pretreated for 1 h with each compound (0.5-5 μM). LPS treated BV2 cells were used as a positive control. After incubation for 24 h, nitric oxide production was measured using the Griess assay and concentrations determined by comparison to a standard curve. 
         FIG. 9  shows absorption and emission spectra of BDNAC325, BDN 325-7 and BDNCA325, which were measured at the concentration of 200 μM in DMSO. 
         FIG. 10 a -10 c    show BV2 microglia that were co-stained with Mitotracker and BDNCA1H5 ( FIG. 10 a   ) or Lysotracker and BDNCA1 H5 ( FIG. 10 b   ). The compound shows good co-localization with Mitotracker.  FIG. 10 c    shows an SDS-PAGE gel run with BV2 cell lysate extracts with the target band appearing as a strong fluorescence signal at the 25 kDa marker size (left). GAPDH was used as a loading control and COX IV was used as mitochondrial loading control to show that the signal is coming predominantly from the mitochondrial fraction. 
         FIGS. 11 a  and 11 b    show BV2 microglia activated with LPS showing brighter BDNCA1 H5 staining intensity as compared to their non-activated counterparts. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A description of example embodiments of the invention follows. 
     A novel class of dyes having a core structure of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is described herein. BODIPY fluorescent dyes are known in the art for outstanding photophysical properties as a fluorescent scaffold, such as high photostability, high fluorescent quantum yield, high extinction coefficient, and narrow emission bandwidth (Karolin, J.; Johansson, L.; Strandberg, L.; Ny, T.  J. Am. Chem. Soc.  1994, 116, 7801-7806). The synthesis of a BODIPY-based library on solid support, and the subsequent discovery of pancreatic islet in vivo sensor and microglia sensor is disclosed. 
     Synthesis of BODIPY Fluorescent Sensors. 
     Scheme I, shown below, depicts a general synthetic scheme for the compounds of the invention. 
     
       
         
         
             
             
         
       
     
     One embodiment of the invention are compounds represented by structural Formula (I) or pharmaceutically acceptable salts thereof: 
                         
wherein:
     R 1  is hydrogen or —COR 3 ;   R 2  is (C 6 -C 16 )aryl, (C 3 -C 10 )heteroaryl, (C 1 -C 6 )alkyl, (C 1 -C 6 )cycloalkyl, (C 2 -C 6 )alkenyl, or C≡CH;   R 2  is optionally substituted with 1-5 substituents independently selected from (C 1 -C 6 )alkyl, halogen, amino, cyano, —COOH, halo(C 1 -C 6 )alkyl, hydroxy(C 0 -C 6 )alkyl, (C 6 -C 10 )aryl, (C 3 -C 10 )heteroaryl, halo(C 6 -C 10 )aryl, hydroxy(C 6 -C 10 )aryl, (C 1 -C 6 )alkoxy, halo(C 1 -C 6 )alkoxy, (C 6 -C 16 )aryloxy, (C 3 -C 8 )cycloalkyl, halo(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, (C 1 -C 6 )alkoxy(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, nitro, (C 0 -C 6 )alkyl(C 6 -C 10 )aryl(C 0 -C 6 )alkoxy, (C 5 -C 10 )heterocycle, —OCHF 2 , —OCF 3 , —SCF 3 , —OBn, cyano(C 1 -C 6 )alkylene, (C 1 -C 6 )alkoxyamino, (C 6 -C 10 )aryl(C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 1 -C 6 )alkoxy, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 6 -C 10 )aryl, —N((C 0 -C 6 )alkyl)((C 1 -C 6 )alkyl), —N((C 1 -C 6 )alkyleneOH)((C 1 -C 6 )alkyleneOH), —N((C 0 -C 6 )alkyl)((C 1 -C 6 )alkyleneOH), —N((C 1 -C 6 )alkyleneOCO(C 1 -C 6 )alkyl)((C 1 -C 6 )alkyleneOCO(C 1 -C 6 )alkyl), —NCO(C 1 -C 6 )alkyl, —NPh 2 , —OPh(halogen) 0-3 , —OPhO(C 1 -C 6 )alkyl, —OPhO(C 1 -C 6 )alkyl, —OCO(C 1 -C 6 )alkyl, —OCO(C 1 -C 6 )alkoxy, —O(C 1 -C 6 )alkyl(C 6 -C 10 )aryl, —O(C 2 -C 6 )alkenyl, —O(C 2 -C 6 )alkyleneN(CH 3 ) 2 , (C 0 -C 6 )alkylCOO(C 1 -C 6 )alkyl, —B(OH) 2  or —S(C 1 -C 6 )alkyl; and wherein any of the substituents selected from (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryl, (C 6 -C 16 )aryloxy or (C 5 -C 10 )heteroaryl is further optionally substituted with 1-4 substituents selected from halogen, (C 1 -C 6 )alkyl, halo(C 1 -C 6 )alkyl, amino, nitro, cyano, hydroxy(C 0 -C 6 )alkyl, (C 1 -C 6 )alkoxy, —COO(C 0 -C 6 )alkyl, or —CHO; and   R 3  is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 6 -C 10 )aryl or (C 5 -C 10 )heteroaryl, wherein R 3  is optionally substituted with 1-4 substituents independently selected from halogen, amino, cyano or hydroxyl.   

     In another embodiment, compounds of the present invention are represented by Formula (I), wherein:
     R 1  is hydrogen or —COR 3 ;   R 2  is (C 6 -C 10 )aryl, (C 5 -C 10 )heteroaryl or (C 1 -C 6 )alkyl;   R 2  is optionally substituted with 1-4 substituents independently selected from (C 1 -C 6 )alkyl, halogen, amino, cyano, halo(C 1 -C 6 )alkyl, hydroxy(C 0 -C 6 )alkyl, (C 6 -C 10 )aryl, (C 5 -C 10 )heteroaryl, halo(C 6 -C 10 )aryl, hydroxy(C 6 -C 10 )aryl, (C 1 -C 6 )alkoxy, halo(C 1 -C 6 )alkoxy, (C 3 -C 8 )cycloalkyl, halo(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, (C 1 -C 6 )alkoxy(C 6 -C 10 )aryl(C 1 -C 6 )alkoxy, nitro, (C 0 -C 6 )alkyl(C 6 -C 10 )aryl(C 0 -C 6 )alkoxy, (C 5 -C 10 )heterocycle, —OCHF 2 , —OCF 3 , —OBn, cyano(C 1 -C 6 )alkylene, (C 1 -C 6 )alkoxyamino, (C 6 -C 10 )aryl(C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 1 -C 6 )alkoxy, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkenyl(C 6 -C 10 )aryl, —N(C 0 -C 6 )alkyl(C 1 -C 6 )alkyl, —N(C 1 -C 6 )alkyleneOH(C 1 -C 6 )alkyleneOH, —N(C 0 -C 6 )alkyl(C 1 -C 6 )alkylOH, —NPh 2 , —OPh(halogen) 0-3 , —OPhO(C 1 -C 6 )alkyl, —OPhO(C 1 -C 6 )alkyl, —OCO(C 1 -C 6 )alkyl, —O(C 1 -C 6 )alkyl(C 6 -C 10 )aryl, —O(C 2 -C 6 )alkenyl, —O(C 2 -C 6 )alkyleneN(CH 3 ) 2 , (C 0 -C 6 )alkylCOO(C 1 -C 6 )alkyl, —B(OH) 2  or —S(C 1 -C 6 )alkyl; and wherein any of the substituents selected from (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryl, or (C 5 -C 10 )heteroaryl is further optionally substituted with 1-4 substituents selected from halogen, (C 1 -C 6 )alkyl, amino, nitro, cyano, hydroxy(C 0 -C 6 )alkyl, (C 1 -C 6 )alkoxy, or —HC═O; and   R 3  is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 6 -C 10 )aryl or (C 5 -C 10 )heteroaryl, wherein R 3  is optionally substituted with 1-4 substituents independently selected from halogen, amino, cyano or hydroxyl.   

     In a preferred embodiment of the invention, R 2  is phenyl, optionally substituted to 1-4 substituents selected from (C 1 -C 6 )alkyl and (C 1 -C 6 )alkoxy. In a more preferred embodiment, a compound of the invention is a compound of formula (II): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, a compound of formula (III) is disclosed: 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment, a compound of the invention is a compound of formula (IV): 
     
       
         
         
             
             
         
       
     
     In another preferred embodiment of the invention, the compound has the formula (V): 
                         
Alternately, the compound of formula (V) may be chlorinated, represented by formula (VI).
 
     
       
         
         
             
             
         
       
     
     Further, any combination of substituents set forth in Table 2 are embodiments of the present invention. 
     Also described herein are methods of synthesizing the BODIPY fluorescent sensors of the present invention. The invention provides solid-phase methods of synthesizing compounds of Formula (I), which comprises the steps:
     (a) reacting a compound of structural Formula (VII):   

     
       
         
         
             
             
         
       
         
         
           
             with a base and an aldehyde, such that the activated C 3 -methyl group of Formula (VII) is modified in a solid-phase Knoevenagel-type reaction to produce a compound of Formula VIII: 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             wherein R 2  is defined as it is in Formula (I); 
           
         
         b) removing the solid support resin from the compound of structural Formula (VIII) to generate a BODIPY structure of Formula (IX): 
       
    
     
       
         
         
             
             
         
       
         
         
           
             wherein R 2  is defined as it is in Formula (I); 
           
         
         c) optionally reacting the compound of structural Formula (IX) with an acid chloride of the formula R 1 (CO)Cl to generate a compound of structural Formula (I), wherein R 1  is defined as in Formula (I). 
       
    
     In some embodiments of the invention, the solid support resin is 2-chlorotrityl polystyrene resin (CT-PS). The base in the Knoevenagel-type reaction is an amine base, such as pyrrole, piperidine, triethylamine, pyrrolidine, or pyridine. In a preferred embodiment of the invention, the base is pyrrolidine. Furthermore, the solvent that the Knoevenagel reaction is carried out in is N-Methyl-2-pyrrolidone, n-butanol, or any combination thereof. In some embodiments of the invention, the CT-PS resin is removed with an organic acid (e.g. R—COOH) or a mineral acid. In a preferred embodiment of the invention, the removal of the CT-PS resin is carried out with trifluoroacetic acid, and is carried out in a chlorinated solvent such as chloroform, methylene chloride, dichloroethane, and most preferably, methylene chloride. 
     The BODIPY structure of Formula (IX) is a compound of structural formula (I) and therefore a novel compound of the invention. The aniline moiety of Formula (IX) may be further substituted by reaction with an acid chloride (e.g. R 1 COCl), to install the R 1  substituent of Formula (I). A compound of Formula (IX) is optionally reacted with an acid chloride to modify the aniline moiety. The aniline substitution reaction is carried out with aqueous base solution, preferably NaHCO 3 . 
     Another embodiment of the invention are methods for detecting beta-cells using image based screening, comprising: a) contacting a sample comprising pancreas cells with a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of Formula (I) is described above; b) incubating the sample and the compound of step a) together for a period of time sufficient to stain the cells; and c) analyzing the incubated dye-stained cells by spectroscopy to detect a fluorescence signal, wherein the presence of a fluorescence signal indicates the presence of beta-cells. 
     In one embodiment of the invention, the sample that is used in the method for detecting beta-cells is isolated mouse pancreas. In a preferred embodiment of the invention, the sample is a pancreatic tissue section or isolated pancreas islets. In another embodiment of the invention, the sample is thought to comprise beta-cells. The methods for detecting beta-cells described herein are selective for the detection of beta-cells over other cell types. The methods for detecting beta-cells are used in an in vitro application. Alternately, the methods of the invention are used in an in vivo application. When the methods of detecting beta-cells are for use in vitro analysis, a preferred embodiment of the invention utilizes a BODIPY dye of Formula (II), called BDNCA 325. When the methods of detecting beta-cells are for use in vivo analysis, a preferred embodiment of the invention utilizes a BODIPY dye of Formula (III), called BDNAC 325. Further, the beta-cells are preferably pancreatic islet cells. In some embodiments of the invention, the period of time in which the sample and BODIPY dye is incubated together is from about 1 hour to about 48 hours. In a preferred embodiment of the invention, the period of time in which the sample and BODIPY dye is incubated together is from about 1 hour to about 24 hours. In some embodiments of the invention, the incubated mixture is analyzed by fluorescence microscope. The methods disclosed in the present invention analyze a sample by a fluorescent signal, wherein the presence of the signal is indicative of the presence of the beta-cells. 
     According to one embodiment of the invention, cells of interest can be screened using a negative control cell type to determine the selectivity of the specific compounds of the invention to cells that are being detected. Such negative control for detection of the beta-cell is, for example, an acinar cell. 
     Another embodiment of the invention provides methods for detecting microglia cells, comprising: a) contacting a sample comprising cells with a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof; b) incubating the sample and compound of step a) together for a period of time sufficient to stain the cells; and c) analyzing the incubated stained cells by spectroscopy, wherein the presence of a fluorescence signal is indicative of the presence of the microglia cells 
     In one embodiment of the invention, the sample that is used in the methods for detecting microglia cells is a primary neural cell culture or a microglia-derived cell line culture such as BV2 microglia cell line. In another embodiment of the invention, the sample is thought to comprise beta cells. The methods described herein for detecting microglia cells are selective for the detection of microglia cells over primary neural cells and other cell types. Further, BODIPY dye utilized in the methods for detecting microglia cells described herein does not affect cellular function and is not cytotoxic. The methods for detecting microglia cells are for use in vitro, or alternatively in vivo. In one aspect of the invention, the BODIPY dye is modified with a radioactive isotope such as  18 F or  11 C, enabling detection of the compound by PET analysis in vivo applications. A preferred compound of Formula (I) used in the detection of microglia cells is a BODIPY dye of Formula (VI), called BDNCA 164 or BDNCA1 H5. In a preferred embodiment of the invention, the sample and compound are incubated together for about one hour. In another aspect of the invention, the incubated mixture is washed in fresh media for about one hour. In some embodiments of the invention, the incubated mixture is analyzed by fluorescence microscope or flow cytometry. The methods disclosed in the present invention analyze a sample by a fluorescent signal, wherein the presence of the signal is indicative of the presence of microglia cells. 
     The negative control for microglia screening is, for example, neuronal stem cell, primary neuron cell or astrocyte cell. In the preferred embodiment the dye is BDNCA164. In one aspect of the invention, the BODIPY dye staining is localized in the mitochondria of the microglia cells. In a further embodiment of the invention, the method of detecting microglia cells can discriminate between resting and activated microglia cells. Utilizing BODIPY dye BDNCA 164, activated microglia cells are visualized with fluorescence intensity about 2-times greater than resting microglia cells. In some embodiments of the invention, the fluorescence intensity is measured by fluorescence microscopy or by flow cytometry. 
     Another embodiment of the invention provides methods for fluorescence imaging of pancreatic islet cells to determine health status of the pancreatic islet cells, comprising: a) contacting the pancreatic islet cells with a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof; b) incubating the cells and the compound of step a) together for a period of time sufficient to stain the cells; c) analyzing the incubated stained pancreatic islet cells by fluorescent microscopy to detect a fluorescence signal; and d) comparing the signal from step c) to a signal from reference sample of healthy pancreatic islet cells, to determine health status of the pancreatic islet cells. 
     According to one embodiment of the invention, healthy pancreatic islet cells are preferentially stained with the fluorescent BODIPY dye compounds of the invention. Without being bound to theory, it is believed that healthy pancreatic islet cells are preferentially stained because the healthy pancreatic islet cells are surrounded by beta cells that are stainable, whereas the beta cells are broken down around the diseased cells and thereby the diseased cells are not stained. The methods for determining health status by imaging of pancreatic islets are used in an in vitro application. Alternately, the methods are used in an in vivo application. When the methods of imaging pancreatic islets are for use in an in vitro application, a preferred embodiment of the invention utilizes a BODIPY dye of Formula (II), called BDNCA 325. When the methods of detecting beta-cells are for use in an in vivo application, a preferred embodiment of the invention utilizes a BODIPY dye of Formula (III), called BDNAC 325, or PiY. 
     In some embodiments of the invention, the period of time in which the sample and the BODIPY dye is incubated together is from about 0.5 hours to about 4 hours. Preferably, the incubation time is from about 0.5 hour to about 1 hour. Health status of the pancreatic islets can be determined by comparing the fluorescence of the test sample to a reference sample. In one embodiment, the reference sample is a sample of healthy pancreatic cells. The signal characteristics that are compared can be, but are not limited to, fluorescence intensity, staining localization, selectivity among cell type and tissue, or quantification by signal intensity. A fluorescence signal and its signal characteristics are ascertained through spectroscopy or fluorescence microscopy using a fluorometer, UV/VIS spectrometer, Gemini XS fluorescence plate reader, flow cytometry or a confocal microscope. 
     The methods described herein can be performed in vitro or in vivo to ascertain, for example, the status or location of target cells, for example, the in vivo methods can be used to probe for pancreatic transplants in a patient or study microglia related inflammation. The methods can be performed on a mammal, preferably human. 
     Definitions 
     “Alkyl” means a saturated aliphatic branched or straight-chain monovalent hydrocarbon radicals, typically C 1 -C 10 , preferably C 1 -C 6 . “(C 1 -C 6 ) alkyl” means a radical having from 1-6 carbon atoms in a linear or branched arrangement. “(C 1 -C 6 )alkyl” includes methyl, ethyl, propyl, butyl, tert-butyl, pentyl and hexyl. 
     “Alkylene” means a saturated aliphatic straight-chain divalent hydrocarbon radical. Thus, “(C 1 -C 6 )alkylene” means a divalent saturated aliphatic radical having from 1-6 carbon atoms in a linear arrangement. “(C 1 -C 6 )alkylene” includes methylene, ethylene, propylene, butylene, pentylene and hexylene. 
     “Heterocycle” means a saturated or partially unsaturated (3-7 membered) monocyclic heterocyclic ring containing one nitrogen atom and optionally 1 additional heteroatom independently selected from N, O or S. When one heteroatom is S, it can be optionally mono- or di-oxygenated (i.e., —S(O)— or —S(O) 2 —). Examples of monocyclic heterocycle include, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, hexahydropyrimidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine, thiomorpholine 1,1-dioxide, tetrahydro-2H-1,2-thiazine, tetrahydro-2H-1,2-thiazine 1,1-dioxide, isothiazolidine, or isothiazolidine 1,1-dioxide. 
     “Cycloalkyl” means saturated aliphatic cyclic hydrocarbon ring. Thus, “C 3 -C 7  cycloalkyl” means (3-7 membered) saturated aliphatic cyclic hydrocarbon ring. C 3 -C 7  cycloalkyl includes, but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Preferably, cycloalkyl is C 1 -C 6  cycloalkyl. 
     The term “alkoxy” means —O-alkyl; “hydroxyalkyl” means alkyl substituted with hydroxy; “aralkyl” means alkyl substituted with an aryl group; “alkoxyalkyl” mean alkyl substituted with an alkoxy group; “alkylamine” means amine substituted with an alkyl group; “cycloalkylalkyl” means alkyl substituted with cycloalkyl; “dialkylamine” means amine substituted with two alkyl groups; “alkylcarbonyl” means —C(O)-A*, wherein A* is alkyl; “alkoxycarbonyl” means —C(O)—OA*, wherein A* is alkyl; and where alkyl is as defined above. Alkoxy is preferably O(C 1 -C 6 )alkyl and includes methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy. 
     “Cycloalkoxy” means an cycloalkyl-O— group wherein the cycloalkyl is as defined above. Exemplary (C 3 -C 7 )cycloalkyloxy groups include cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy and cycloheptoxy. 
     “Hetero” refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom selected from N, S, and O. A hetero ring system may have 1 or 2 carbon atom members replaced by a heteroatom. 
     “Halogen” and “halo” are interchangeably used herein and each refers to fluorine, chlorine, bromine, or iodine. 
     “Cyano” means —C≡N. 
     “Nitro” means —NO 2 . 
     As used herein, an amino group may be a primary (—NH 2 ), secondary (—NHR x ), or tertiary (—NR x R y ), wherein R x  and R y  may be any of the optionally substituted alkyls described above. An amino group may include cyclic amino groups such as piperidine and pyrrolidine. 
     The terms “haloalkyl”, “halocycloalkyl” and “haloalkoxy” mean alkyl, cycloalkyl, or alkoxy, as the case may be, substituted with one or more halogen atoms. The term “halogen” means F, Cl, Br or I. Preferably the halogen in a haloalkyl or haloalkoxy is F. 
     The term “acyl group” means —C(O)B*, wherein B* is an optionally substituted alkyl group or aryl group (e.g., optionally substituted phenyl). 
     An “alkylene group” is represented by —[CH 2 ] z —, wherein z is a positive integer, preferably from one to eight, more preferably from one to four. 
     An “alkenylene group” is an alkylene in which at least a pair of adjacent methylenes are replaced with —CH═CH—. 
     The term “(C 6 -C 16 )aryl” used alone or as part of a larger moiety as in “arylalkyl”, “arylalkoxy”, “aryloxy”, or “aryloxyalkyl”, means carbocyclic aromatic rings. The term “carbocyclic aromatic group” may be used interchangeably with the terms “aryl”, “aryl ring” “carbocyclic aromatic ring”, “aryl group” and “carbocyclic aromatic group”. An aryl group typically has 6-16 ring atoms. A “substituted aryl group” is substituted at any one or more substitutable ring atom. The term “C 6 -C 16  aryl” as used herein means a monocyclic, bicyclic or tricyclic carbocyclic ring system containing from 6 to 16 carbon atoms and includes phenyl (Ph), naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. 
     The term benzyl (Bn) refers to —CH 2 Ph. 
     The term “heteroaryl”, “heteroaromatic”, “heteroaryl ring”, “heteroaryl group” and “heteroaromatic group”, used alone or as part of a larger moiety as in “heteroarylalkyl” or “heteroarylalkoxy”, refers to aromatic ring groups having five to fourteen total ring atoms selected from carbon and at least one (typically 1-4, more typically 1 or 2) heteroatoms (e.g., oxygen, nitrogen or sulfur). They include monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. The term “5-14 membered heteroaryl” as used herein means a monocyclic, bicyclic or tricyclic ring system containing one or two aromatic rings and from 5 to 14 total atoms of which, unless otherwise specified, one, two, three, four or five are heteroatoms independently selected from N, NH, N(C 1-6 alkyl), O and S. (C 3 -C 10 )heteroaryl includes furyl, thiophenyl, pyridinyl, pyrrolyl, imidazolyl, and in preferred embodiments of the invention, heteroaryl is (C 3 -C 10 )heteroaryl. 
     The term “2-4 member polycyclyl” is a cyclic compound with 2-4 hydrocarbon loop or ring structures (e.g., benzene rings). The term generally includes all polycyclic aromatic compounds, including the polycyclic aromatic hydrocarbons, the heterocyclic aromatic compounds containing sulfur, nitrogen, oxygen, or another non-carbon atoms, and substituted derivatives of these. 
     The term “Alkenyl” means a straight or branched hydrocarbon radical having a specified number of carbon atoms and includes at least one double bond. The (C 6 -C 10 )aryl(C 2 -C 6 )alkenyl group connects to the remainder of the molecule through the (C 2 -C 6 )alkenyl portion of (C 6 -C 10 )aryl(C 2 -C 6 )alkenyl. 
     Another embodiment of the present invention are a pharmaceutical compositions comprising one or more pharmaceutically acceptable carrier and/or diluent and a compound disclosed herein or a pharmaceutically acceptable salt thereof. 
     “Pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” means non-therapeutic components that are of sufficient purity and quality for use in the formulation of a composition of the invention that, when appropriately administered to an animal or human, typically do not produce an adverse reaction, and that are used as a vehicle for a drug substance (i.e., a compound of the present invention). 
     Pharmaceutically acceptable salts of the compounds of the present invention are also included. For example, an acid salt of a compound of the present invention containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts. 
     Salts of the compounds of the present invention containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine. 
     “CT-PS resin” is a resin used in solid phase synthesis. Specifically, it is 2-chlorotrityl-polystyrene. 
     A “cellular extract” is lysed cells from which insoluble matter has been removed via centrifugation. 
     A “tissue section” is a portion of tissue suitable for analysis. A tissue section can refer to a single tissue section or a plurality of tissue sections. 
     As used herein, “spectroscopy” encompasses any method by which matter reacts with radiated energy. This includes, but is in no way limited to, microscopy, fluorescence microscopy, UV/Vis spectrometry, and flow cytometry. 
     EXAMPLES 
     The following examples are provided to illustrate one or more preferred embodiments of the invention, but are not limited embodiments thereof. Numerous variations can be made to the following examples that lie within the scope of the invention. 
     Example 1 
     Synthetic Protocol for the BODIPY Dye Compounds of the Invention 
     Using inexpensive starting materials (pyrrole and nitrobenzoyl chloride), Compound 1 was prepared according to the following method: 
                         
(4-nitrophenyl)(1H-pyrrol-2-yl)methanone (1). CH 3 MgBr (3M) in diethylether (30 mL, 90 mmol) was added by dropwise to the solution of pyrrole (6.95 mL, 100 mmol) in THF (150 mL) at −78° C. and continued for 1 h at r.t. 4-nitrobenzoyl chloride (13 g, 70 mmol) in THF (200 mL) was added by dropwise to the solution and stirring was continued for 3 hours at r.t. The mixture was poured into saturated aqueous NH 4 Cl, and the precipitate dissolved in DCM and added to the aqueous solution. The organic layer was washed with water, dried over MgSO 4  and concentrated under vacuum to give an oily product. Compound 1 was isolated by chromatography on silica gel flash column eluting with EtOAc/Hex to furnish a yellow solid (12.4 g, 81%).  1 H-NMR (CDCl 3 ) δ 10.01 (bs, 1H, NH), 8.34 (d, J=8.4, 2H), 8.03 (d, J=8.7, 2H), 7.24 (bs, 1H), 6.86 (bs, 1H), 6.38 (bs, 1H);  13 C-NMR (CDCl 3 ) δ 182.51, 149.61, 143.62, 130.57, 129.74, 126.67, 123.55, 120.37, 111.69. ESI-MS m/z (M+H) calc&#39;d: 217.0 found 216.9.
 
     Further condensation with 2,4-dimethylpyrrole and InCl 3 , followed by in situ addition of the BF 3 .OEt 2  yielded BODIPY derivative 2. 
                         
4,4-Difluoro-1,3-dimethyl-8-(4-nitrophenyl)-4-bora-3a,4a-diaza-s-indacene (2). A solution of compound 1 (8.64 g, 40 mmol) in THF/MeOH (110 mL, 10:1) was treated with NaBH 4  (3.03 g, 80 mmol) at r.t. for 1 h. The reaction mixture was poured in a mixture of saturated aqueous NH 4 Cl (100 mL) and EtOAc (200 mL). The organic phase was separated, washed with water, dried (Na 2 SO 4 ), and concentrated to dryness, affording crude yellow oil (8.38 g, 38.8 mmol). The crude reduced alcohol product was used directly without purification due to the instability.
 
     A mixture of the resulting residue and 2,4-dimethylpyrrole (4.02 mL, 39 mmol) was dissolved in DCM (100 mL) and treated with InCl 3 (858 mg, 3.88 mmol) at room temperature. After 3 hours, the reaction mixture was quenched with triethylamine (557 μL, 4 mmol). The organic layer was concentrated, and silica gel filtered with DCM, affording crude compound of coupled dipyrrole (7.37 g, 25 mmol), which was used directly in next step. 
     To a solution of dipyrrole (7.37 g, 25 mmol) in toluene (100 mL) was added DDQ (5.7 g, 25 mmol) in DCM (20 mL). The mixture was stirred 15 min at r.t. A solution of reaction mixture was cooled under N 2  to 0° C. Triethylamine (21 mL, 150 mmol) was added for neutralization. After 2 min of stirring, BF 3 .OEt 2  (31 mL, 250 mmol) was added dropwise, over 10 min. Thereafter, the mixture was stirred for 8 h at rt. The reaction solution was filtered through short panel of silica gel with DCM-Hex (1:2) solution, affording crude compound 2. Compound 2 was purified by column chromatography on silica after removal of reaction solvent (8.21 g, 24.1 mmol). 
       1 H-NMR (CDCl 3 ) δ 8.38 (d, J=8.7, 2H), 7.71 (s, 1H), 7.57 (d, J=8.4, 2H), 6.39 (s, 1H), 6.30 (d, J=3.3, 1H), 6.18 (s, 1H), 2.64 (s, 3H), 1.49 (s, 3H);  13 C-NMR (CDCl 3 ) δ 163.58, 148.59, 146.43, 140.67, 139.87, 139.67, 133.78, 130.07, 128.79, 126.69, 123.93, 123.74, 116.67, 29.67, 15.25, 15.15. ESI-MS m/z (M+H) calc&#39;d: 342.1, found: 342.0. 
     The nitro group of 2 was converted to a primary amine under reductive conditions. 
                         
4,4-Difluoro-1,3-dimethyl-8-(4-aminoophenyl)-4-bora-3a,4a-diaza-s-indacene (3). A solution of compound 2 (8.21 g, 24.1 mmol) in EtOH (100 mL) was purged with N 2  for 10 min. Hydrazine monohydrate (5.81 mL) and 10% Pd/C (100 mg) were added. The mixture was refluxed under N 2  for 2 hours. Then Pd/C was removed under celite filtration. After evaporation of the solvent, the residue was dry-loaded onto a silica gel flash column and eluted using EtOAc/Hex to afford an orange solid (5.85 g, 18.8 mmol).  1 H-NMR (CDCl 3 ) δ 7.65 (s, 1H), 7.13 (d, J=8.4, 2H), 6.75 (d, J=8.1, 2H), 6.52 (d, J=3.3, 1H), 6.37 (d, J=1.8, 1H), 6.12 (s, 1H), 2.61 (s, 3H), 1.67 (s, 3H);  13 C-NMR (CDCl 3 ) δ 161.00, 147.64, 146.70, 144.44, 138.02, 135.01, 133.69, 130.39, 126.92, 123.77, 122.88, 115.74, 114.57, 29.65, 15.43, 15.03: ESI-MS m/z (M+H) calc&#39;d: 312.1, found: 312.4.
 
Loading of BODIPY on 2-Chlorotrityl Resin.
 
     The aminophenyl BODIPY (3) was loaded to 2-chlorotrityl resin. In DCM solvent aminophenyl BODIPY compound 3 and pyridine (5 equiv) were diluted and added to pre-swelled 3 equiv. of 2-chlorotrityl resin (1 mmol/g) and kept shaking 12 h. 20 equiv. methanol was added for quenching of extra chloride on resin. After 1 h shaking for quenching, resin was washed with DMF, MeOH, DCM and dried with high vacuum pump. 
     Condensation Reaction with Aldehyde and Cleavage. 
     The red bead (150 mg, 50 μmole) was placed in 20 mL capped vial swell with 4 mL of NMP/n-BuOH (4/1) and operated in microwave chamber with 300 W power for 2 minutes, monitoring the color change it was repeated until black color was appeared. The black color resin was washed with DMF, MeOH, DCM and directly cleaved by 0.5% of TFA in DCM solution for 10 minutes and repeated twice. Generated compounds were purified on silica packed syringe with EtOAc/Hex solvent. 
     Acylation of BDN Compounds: 
     Purified aniline BODIPY compound was diluted in DCM/ACN (2/1), 10 μL of NaHCO 3  saturated solution was added and acid chloride (5 equiv.) was added immediately. The organic layer was washed with NaHCO 3  saturated solution twice. 
                         
Characterization of BDNAC 325.  1 H-NMR (CDCl 3 ) δ 7.72 (m, 6H), 7.60 (d, J=2.4 Hz, 1H), 7.42 (d, J=2.4 Hz, 1H), 7.30 (s, 1H) 6.81 (s, 1H), 6.43 (m, 2H), 3.8 (s, 3H), 2.27 (s, 3H), 1.45 (s, 9H), 1.39 (s, 9H);  13 C-NMR (CDCl 3 ) δ 166.16, 164.25, 162.80, 157.87, 156.88, 151.60, 146.14, 145.84, 144.99, 142.19, 139.31, 138.02, 136.75, 135.87, 129.80, 129.63, 129.16, 127.46, 127.11, 126.34, 126.31, 126.24, 122.80, 119.49, 119.21, 118.75, 112.80, 104.96, 63.31, 60.39, 35.23, 34.64, 31.44, 30.93, 29.67, 24.67, 21.81, 15.50, 15.47, 14.18, 10.21.; ESI-MS m/z (M+H) calc&#39;d: 584.31, found 584.30.
 
                         
Characterization of BDN 325-7.  1 H-NMR (CDCl 3 ) δ 7.72 (m, 6H), 7.57 (d, J=2.1 Hz, 1H), 7.38 (d, J=2.1 Hz, 1H), 7.32 (d, J=8.7 Hz, 2H), 6.77 (s, 1H), 6.45 (m, 1H), 6.39 (m, 2H), 6.30 (m, 1H), 5.78 (dd, J=1.2, 10.2, 1H), 3.78 (s, 3H), 1.64 (s, 3H), 1.41 (s, 9H), 1.35 (s, 9H);  13 C-NMR (CDCl 3 ) 163.73, 159.93, 158.67, 156.91, 151.28, 146.16, 145.82, 143.42, 142.24, 141.10, 139.09, 138.04, 136.87, 135.04, 130.879, 129.95, 129.86, 129.13, 128.40, 126.37, 126.25, 122.79, 119.48, 119.45, 119.30, 118.72, 118.69, 116.096, 63.33, 35.24, 34.64, 31.44, 30.94, 29.68, 15.53, 14.10, ESI-MS m/z (M+H) calc&#39;d: 596.32, found 596.30.
 
                         
Characterization of BDNCA 325.  1 H-NMR (CDCl 3 ) δ 8.40 (s, 1H), 7.72 (m, 5H), 7.57 (d, J=2.4 Hz, 1H), 7.39 (m, 3H), 6.78 (s, 1H), 6.40 (bs, 2H), 4.2 (s, 2H), 3.79 (s, 3H), 1.64 (s, 3H), 1.42 (s, 9H), 1.36 (s, 9H);  13 C-NMR (CDCl 3 ) 163.98, 158.85, 156.92, 146.18, 145.69, 142.21, 140.66, 138.20, 137.83, 136.95, 134.87, 130.92, 129.99, 129.16, 126.38, 126.13, 122.84, 119.64, 119.34, 118.74, 116.09, 104.96, 102.33, 101.02, 63.35, 42.88, 35.26, 34.66, 31.46, 30.96, 29.69, 15.50, ESI-MS m/z (M+H) calc&#39;d: 618.18, found 618.30
 
                         
Characterization of BDNCA 164.  1 H-NMR (CD 3 OD+CDCl 3 ) δ 8.36 (s, 1H), 7.71 (m, 3H), 7.55 (d, J=16.0 Hz, 1H), 7.39 (d, J=8.5 Hz, 2H), 7.33 (d, J=16.0 Hz, 1H), 7.17 (m, 2H), 6.88 (d, J=9.0 Hz, 1H), 6.72 (s, 1H), 6.38 (bs, 2H), 4.25 (s, 2H), 4.18 (m, 2H), 3.92 (s, 3H), 1.63 (s, 3H), 1.50 (t, J=7 Hz, 3H),  13 C-NMR (CDCl 3 ) δ 165.35, 158.33, 150.99, 148.28, 145.69, 140.13, 138.52, 136.95, 134.65, 129.95, 129.49, 128.76, 122.30, 119.42, 119.12, 115.96, 111.15, 110.86, 64.30, 55.57, 42.66, 29.31, 15.01, 14.22; ESI-MS m/z (M+H) calc&#39;d: 550.18, found 550.20, 530.20 (M−F)
 
                         
Characterization of BDNAC 164;  1 H-NMR (CD 3 OD+CDCl 3 ) δ 8.46 (s, 1H), 7.76 (m, 3H), 7.65 (d, J=16.0 Hz, 1H), 7.43 (d, J=8.5 Hz, 2H), 7.32 (d, J=16.0 Hz, 1H), 7.19 (m, 2H), 6.89 (d, J=9.0 Hz, 1H), 6.75 (s, 1H), 6.39 (bs, 2H), 2H), 4.20 (m, 2H), 3.95 (s, 3H), 2.04 (s, 3H), 1.71 (s, 3H), 1.49 (t, J=7 Hz, 3H),  13 C-NMR (CDCl 3 ) δ 165.45, 159.33, 151.99, 147.28, 146.69, 140.33, 138.55, 136.98, 134.65, 129.95, 129.49, 128.76, 123.30, 119.52, 119.82, 114.96, 112.15, 110.86, 64.30, 55.58, 42.66, 29.51, 15.01, 14.22; ESI-MS m/z (M+H) calc&#39;d: 516.22, found 516.20.
 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Spectral information summary of islet probes 
               
               
                 Spectral Information 
               
            
           
           
               
               
               
               
            
               
                   
                 BDNAC 325 
                 BDN 325-7 
                 BDNCA 325 
               
               
                   
               
               
                 Absorption Max 
                 558 nm 
                 558 nm 
                 558 nm 
               
               
                 Emission Max 
                 585 nm 
                 586 nm 
                 585 nm 
               
               
                 Quantum Yield 
                   0.05 
                   0.06 
                   0.06 
               
               
                 Extinction Coefficient 
                 27700 
                 27900 
                 27600 
               
               
                   
               
               
                                   
                  
                  
 
               
            
           
         
       
     
     In each of the above structures, BDN, BDNAC and BDNCA, R is derived from commercially available aldehydes. 
     The following aldehydes were used in the preparation of the BDN, BDNCA, and BDNAC libraries. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Aldehyde building blocks for BDN, BDNCA, BDNAC libraries 
               
            
           
           
               
               
               
            
               
                   
                 ID 
                 Structure 
               
               
                   
                   
               
               
                   
                 L1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L2 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L3 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L4 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L5 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L6 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L7 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L23 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L25 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L27 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L29 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L30 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L32 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L34 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L8 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L9 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L11 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L12 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L13 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L14 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L15 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L16 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L17 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L36 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L37 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L38 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L40 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L41 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L42 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L43 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L44 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L45 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L18 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L19 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L20 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L22 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L52 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L53 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L54 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L57 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L46 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L48 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L49 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L51 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L83 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L85 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L88 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L89 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L90 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L62 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L63 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L65 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L67 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L68 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L69 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L70 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L71 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L72 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L91 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L92 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L93 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L94 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L95 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L96 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L97 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L100 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L101 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L73 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L75 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L76 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L77 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L78 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L82 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L111 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L114 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L103 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L105 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L107 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L108 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L109 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L110 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L178 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L179 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L117 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L126 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L128 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L131 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L132 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L134 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L135 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L137 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L180 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L182 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L185 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L186 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L190 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L191 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L192 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L195 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L198 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L139 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L140 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L143 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L144 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L147 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L153 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L163 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L164 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L177 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L199 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L202 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L206 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L208 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L209 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L210 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L218 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L219 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L220 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L223 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L228 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L231 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L236 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L237 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L238 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L239 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L240 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L257 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L259 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L260 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L261 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L262 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L263 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L267 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L268 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L241 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L242 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L243 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L245 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L246 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L249 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L250 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L251 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L252 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L274 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L275 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L277 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L281 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L282 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L283 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L284 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L285 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L289 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L254 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L255 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L256 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L298 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L299 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L300 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L301 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L303 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L304 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L290 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L292 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L294 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L325 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L326 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L331 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L332 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L333 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L335 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L305 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L307 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L308 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L310 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L311 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L314 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L315 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L316 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L317 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L336 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L340 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L341 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L343 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L345 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L346 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L347 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L349 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L350 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L318 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L319 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L320 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L322 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L323 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L368 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L370 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L371 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L372 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L353 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L359 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L363 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L365 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L366 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L417 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L421 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L423 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L425 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L375 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L376 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L377 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L378 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L384 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L392 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L393 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L396 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L397 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L427 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L428 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L429 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L430 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L431 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L433 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L435 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L437 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L440 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L401 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L403 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L404 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L405 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L407 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L409 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L414 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L458 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Optical properties of BDNCA library, BDN series does not have 
               
               
                 fluorescent emission, BDNAC library has same properties as BDNCA. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 BDNCA Code 
                   
                   
                   
                 Qunt. 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Plate  
                 Code 
                 Well  
                 Abs 
                 Flu 
                 Yield  
                 ε 
                 Flu-Abs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 BDNCA- 
                 BDNCA-1 
                 BDNCA1- 
                 564 
                 602 
                 0.14 
                 91,442 
                 38 
               
               
                 1 
                   
                 A2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-2 
                 BDNCA1- 
                 613 
                 weak 
                 weak 
                 27,735 
                   
               
               
                   
                   
                 A3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-3 
                 BDNCA1- 
                 605 
                 weak 
                 weak 
                 73,593 
                   
               
               
                   
                   
                 A4  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-4 
                 BDNCA1- 
                 570  
                 611 
                 0.18 
                 79,908 
                 41 
               
               
                   
                   
                 A5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-5 
                 BDNCA1-  
                 566  
                 589 
                 0.09 
                 39,542 
                 23 
               
               
                   
                   
                 A6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-6 
                 BDNCA1- 
                 536 
                 weak 
                 weak  
                 41,465 
                   
               
               
                   
                   
                 A7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-7 
                 BDNCA1- 
                 570  
                 616 
                 0.19 
                 87,872 
                 46 
               
               
                   
                   
                 A8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-8 
                 BDNCA1- 
                 562 
                 582 
                 0.04 
                 29,108 
                 20 
               
               
                   
                   
                 A9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-9 
                 BDNCA1- 
                 556 
                 575 
                 0.10 
                 47,780 
                 19 
               
               
                   
                   
                 A10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 557 
                 576 
                 0.10 
                 49,428 
                 19 
               
               
                   
                 12 
                 A11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 599 
                   
                   
                 35,149 
                   
               
               
                   
                 14 
                 B2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 562  
                 585 
                 0.13 
                 55,469 
                 23 
               
               
                   
                 16 
                 B3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 627 
                 weak 
                 weak 
                 52,998 
                   
               
               
                   
                 17 
                 B4  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 569  
                 611 
                 0.08 
                 31,304 
                 42 
               
               
                   
                 18 
                 B5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 558  
                 580 
                 0.13 
                 61,236 
                 22 
               
               
                   
                 19 
                 B6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 568  
                 591 
                 0.02 
                 21,968 
                 23 
               
               
                   
                 22 
                 B7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1-  
                 566  
                 594 
                 0.09 
                 32,952 
                 28 
               
               
                   
                 25 
                 B8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 602 
                 weak 
                 weak  
                 15,652 
                   
               
               
                   
                 30 
                 B9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 583  
                 649 
                 0.06 
                 27,735 
                 66 
               
               
                   
                 32 
                 B10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 551  
                 597 
                 0.14 
                 42,288 
                 46 
               
               
                   
                 34 
                 B11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1-  
                 572 
                 610 
                 0.08 
                 30,755 
                 38 
               
               
                   
                 36 
                 C2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 568 
                 weak 
                 weak 
                 23,890 
                   
               
               
                   
                 38 
                 C3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 556  
                 579 
                 0.12 
                 61,785 
                 23 
               
               
                   
                 40 
                 C4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 556  
                 577 
                 0.08 
                 54,645 
                 21 
               
               
                   
                 42 
                 C5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566  
                 596 
                 0.03 
                 25,812 
                 30 
               
               
                   
                 43 
                 C6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 580 
                 weak 
                 weak 
                 12,632 
                   
               
               
                   
                 44 
                 C7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 566  
                 598 
                 0.13 
                 82,929 
                 32 
               
               
                   
                 45 
                 C8  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566  
                 598 
                 0.10 
                 42,563 
                 32 
               
               
                   
                 46 
                 C9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566  
                 597 
                 0.07 
                 44,211 
                 31 
               
               
                   
                 48 
                 C10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 564  
                 581 
                 0.03 
                 20,046 
                 17 
               
               
                   
                 49 
                 C11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 561  
                 582 
                 0.05 
                 21,419 
                 21 
               
               
                   
                 51 
                 D2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 570  
                 621 
                 0.06 
                 31,579 
                 51 
               
               
                   
                 53 
                 D3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1-  
                 562 
                 581 
                 0.07 
                 38,169 
                 19 
               
               
                   
                 54 
                 D4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 581  
                 627 
                 0.08 
                 33,776 
                 46 
               
               
                   
                 61 
                 D5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 570  
                 618 
                 0.08 
                 38,169 
                 48 
               
               
                   
                 62 
                 D6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 572  
                 623 
                 0.07 
                 35,149 
                 51 
               
               
                   
                 63 
                 D7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 593  
                 630 
                 0.04 
                 30,755 
                 37 
               
               
                   
                 65 
                 D8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 542  
                 582 
                 0.12 
                 36,522 
                 40 
               
               
                   
                 67 
                 D9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 553  
                 573 
                 0.05 
                 27,735 
                 20 
               
               
                   
                 68 
                 D10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 577  
                 610 
                 0.09 
                 40,641 
                 33 
               
               
                   
                 69 
                 D11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 587  
                 677 
                 0.04 
                 29,931 
                 90 
               
               
                   
                 70 
                 E2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 573  
                 618 
                 0.02 
                 17,849 
                 45 
               
               
                   
                 72 
                 E3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 564  
                 585 
                 0.04 
                 27,185 
                 21 
               
               
                   
                 73 
                 E4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 562 
                 600 
                 0.09 
                 35,973 
                 38 
               
               
                   
                 75 
                 E5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 574 
                 602 
                 0.07  
                 27,460 
                 28 
               
               
                   
                 76 
                 E6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 559 
                 581 
                 0.09 
                 45,034  
                 22 
               
               
                   
                 77 
                 E7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 596  
                 weak  
                 weak  
                 37,071 
                   
               
               
                   
                 78 
                 E8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 582 
                 632 
                 0.04 
                 18,398 
                 50 
               
               
                   
                 82 
                 E9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 557 
                 577 
                 0.12  
                 40,366 
                 20 
               
               
                   
                 83 
                 E10  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566 
                 584 
                 0.11 
                 27,185 
                 18 
               
               
                   
                 88 
                 E11  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 580 
                 634 
                 0.08  
                 33,501 
                 54 
               
               
                   
                 89 
                 F2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 563 
                 593 
                 0.04  
                 20,046 
                 30 
               
               
                   
                 90 
                 F3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 559  
                 weak 
                 weak 
                 34,050 
                   
               
               
                   
                 91 
                 F4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 555 
                 579 
                 0.01 
                 15,652 
                 24 
               
               
                   
                 92 
                 F5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 594  
                 weak 
                 weak 
                 16,476 
                   
               
               
                   
                 93 
                 F6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 548 
                 662 
                 0.02  
                 23,890 
                 114 
               
               
                   
                 94 
                 F7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 563 
                 575 
                 0.01 
                 24,989 
                 12 
               
               
                   
                 97 
                 F8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 598 
                 680 
                 0.04  
                 28,558 
                 82 
               
               
                   
                 103 
                 F9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 624  
                 weak 
                 weak 
                 56,018 
                   
               
               
                   
                 107 
                 F10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 549 
                 582 
                 0.10  
                 39,542 
                 33 
               
               
                   
                 108 
                 F11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566 
                 596 
                 0.16 
                 56,568  
                 30 
               
               
                   
                 110 
                 G2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 570 
                 599 
                 0.04 
                 18,947  
                 29 
               
               
                   
                 111 
                 G3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 559 
                 584 
                 0.02 
                 14,554  
                 25 
               
               
                   
                 128 
                 G4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 643 
                 weak 
                 weak 
                 18,124 
                   
               
               
                   
                 131 
                 G5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 569 
                 599 
                 0.02 
                 36,796 
                 30 
               
               
                   
                 132 
                 G6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA1- 
                 566  
                 weak 
                 weak  
                 12,082 
                   
               
               
                   
                 134 
                 G7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 577 
                 618 
                 0.07 
                 37,346 
                 41 
               
               
                   
                 135 
                 G8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 564 
                 584 
                 0.10 
                 43,387 
                 20 
               
               
                   
                 137 
                 G9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 568 
                 607 
                 0.09 
                 35,423 
                 39 
               
               
                   
                 139 
                 G10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 566 
                 598 
                 0.07 
                 27,185 
                 32 
               
               
                   
                 140 
                 G11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 555 
                 577 
                 0.13 
                 50,801 
                 22 
               
               
                   
                 143 
                 H2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 585 
                 weak 
                 weak 
                 62,609 
                   
               
               
                   
                 144 
                 H3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 557 
                 578 
                 0.07 
                 23,616 
                 21 
               
               
                   
                 147 
                 H4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 570 
                 615 
                 0.07 
                 31,030 
                 45 
               
               
                   
                 164 
                 H5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 558 
                 581 
                 0.15 
                 73,867 
                 23 
               
               
                   
                 177 
                 H6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 563 
                 589 
                 0.09 
                 61,785 
                 26 
               
               
                   
                 178 
                 H7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 581 
                 646 
                 0.06 
                 29,931 
                 65 
               
               
                   
                 185 
                 H8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 556 
                 576 
                 0.14 
                 61,510 
                 20 
               
               
                   
                 186 
                 H9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 564 
                 589 
                 0.02 
                 17,025 
                 25 
               
               
                   
                 190 
                 H10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA1- 
                 577 
                 630 
                 0.03 
                 21,693 
                 53 
               
               
                   
                 192 
                 H11 
                   
                   
                   
                   
                   
               
               
                 BDNCA- 
                 BDNCA- 
                 BDNCA2- 
                 605 
                 681 
                 0.01 
                 21,968 
                   
               
               
                 II 
                 195 
                 A2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 562 
                 589 
                 0.20 
                 89,245 
                 27 
               
               
                   
                 101 
                 A3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 574 
                 597 
                 0.10 
                 48,879 
                 23 
               
               
                   
                 100 
                 A4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 576 
                 0.11 
                 51,625 
                 20 
               
               
                   
                 20 
                 A5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 561 
                 585 
                 0.13 
                 57,666 
                 24 
               
               
                   
                 202 
                 A6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 559 
                 578 
                 0.04 
                 33,227 
                 19 
               
               
                   
                 231 
                 A7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 587 
                 0.15 
                 76,064 
                 26 
               
               
                   
                 198 
                 A8  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 622 
                 weak 
                 weak 
                 50,801 
                   
               
               
                   
                 96 
                 A9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 586 
                 0.14 
                 61,785 
                 25 
               
               
                   
                 218 
                 A10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 560 
                 578 
                 0.13 
                 49,977 
                 18 
               
               
                   
                 15 
                 A11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 566 
                 589 
                 0.14 
                 52,998 
                 23 
               
               
                   
                 179 
                 B2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 563 
                 583 
                 0.15 
                 59,314 
                 20 
               
               
                   
                 199 
                 B3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 568 
                 589 
                 0.09 
                 50,252 
                 21 
               
               
                   
                 180 
                 B4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 576 
                 0.12 
                 62,334 
                 20 
               
               
                   
                 209 
                 B5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 560 
                 581 
                 0.16 
                 73,043 
                 21 
               
               
                   
                 219 
                 B6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 559 
                 583 
                 0.01 
                 40,915 
                 24 
               
               
                   
                 220 
                 B7  
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 574 
                 618 
                 0.13 
                 44,485 
                 44 
               
               
                   
                 206 
                 B8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 563 
                 578 
                 0.01 
                 37,071 
                 15 
               
               
                   
                 126 
                 B9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 574 
                 580 
                 0.01 
                 31,579 
                 6 
               
               
                   
                 95 
                 B10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 567 
                 597 
                 0.01 
                 25,812 
                 30 
               
               
                   
                 29 
                 B11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 580 
                 0.05 
                 36,247 
                 24 
               
               
                   
                 52 
                 C2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 560 
                 579 
                 0.13 
                 70,572 
                 19 
               
               
                   
                 237 
                 C3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 558 
                 577 
                 0.13 
                 63,707 
                 19 
               
               
                   
                 228 
                 C4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 577 
                 608 
                 0.11 
                 72,220 
                 31 
               
               
                   
                 236 
                 C5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 564 
                 585 
                 0.06 
                 39,817 
                 21 
               
               
                   
                 121 
                 C6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 559 
                 580 
                 0.05 
                 41,739 
                 21 
               
               
                   
                 117 
                 C7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 575 
                 0.06 
                 38,719 
                 19 
               
               
                   
                 105 
                 C8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 558 
                 581 
                 0.12 
                 64,805 
                 23 
               
               
                   
                 208 
                 C9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 554 
                 577 
                 0.11 
                 73,318 
                 23 
               
               
                   
                 27 
                 C10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 608 
                 weak 
                 weak 
                 42,288 
                   
               
               
                   
                 114 
                 C11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 583 
                 weak 
                 weak 
                 59,863 
                   
               
               
                   
                 223 
                 D2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 555 
                 573 
                 0.12 
                 64,531 
                 18 
               
               
                   
                 11 
                 D3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 553 
                 573 
                 0.09 
                 56,842 
                 20 
               
               
                   
                 23 
                 D4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 567 
                 600 
                 0.14 
                 71,121 
                 33 
               
               
                   
                 37 
                 D5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 574 
                 0.06 
                 24,714 
                 18 
               
               
                   
                 191 
                 D6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 575 
                 608 
                 0.05 
                 40,366 
                 33 
               
               
                   
                 238 
                 D7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 583 
                 614 
                 0.08  
                 49,977 
                 31 
               
               
                   
                 85 
                 D8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 579 
                 weak 
                 weak 
                 13,455 
                   
               
               
                   
                 13 
                 D9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 566 
                 595 
                 0.04  
                 24,165 
                 29 
               
               
                   
                 163 
                 D10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 557 
                 575 
                 0.08  
                 37,620 
                 18 
               
               
                   
                 182 
                 D11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 569 
                 606 
                 0.05 
                 23,341 
                 37 
               
               
                   
                 153 
                 E2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 577 
                 0.02 
                 23,066 
                 16 
               
               
                   
                 57 
                 E3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 550 
                 570 
                 0.05 
                 43,661 
                 20 
               
               
                   
                 71 
                 E4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 561 
                 585 
                 0.12 
                 69,474 
                 24 
               
               
                   
                 239 
                 E5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 554 
                 573 
                 0.08 
                 47,506 
                 19 
               
               
                   
                 240 
                 E6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 558 
                 578 
                 0.09 
                 48,879 
                 20 
               
               
                   
                 241 
                 E7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 561 
                 586 
                 0.14 
                 70,847 
                 25 
               
               
                   
                 242 
                 E8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 545 
                 575 
                 0.11 
                 47,506 
                 30 
               
               
                   
                 243 
                 E9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 556 
                 575 
                 0.11  
                 70,023 
                 19 
               
               
                   
                 245 
                 E10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 554 
                 573 
                 0.09 
                 43,387 
                 19 
               
               
                   
                 246 
                 E11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 574 
                 611 
                 0.18 
                 64,256 
                 37 
               
               
                   
                 247 
                 F2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 556 
                 577 
                 0.12 
                 71,945 
                 21 
               
               
                   
                 249 
                 F3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 559 
                 581 
                 0.12  
                 53,547 
                 22 
               
               
                   
                 250 
                 F4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 555 
                 578 
                 0.11 
                 54,096 
                 23 
               
               
                   
                 251 
                 F5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 576 
                 0.08 
                 57,666 
                 20 
               
               
                   
                 252 
                 F6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 559 
                 581 
                 0.09 
                 56,018 
                 22 
               
               
                   
                 254 
                 F7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 575 
                 0.10 
                 62,059 
                 19 
               
               
                   
                 255 
                 F8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 582 
                 0.04 
                 52,449 
                 21 
               
               
                   
                 256 
                 F9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 623 
                 weak 
                 weak 
                 52,174 
                   
               
               
                   
                 257 
                 F10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 582 
                 0.11 
                 49,428 
                 21 
               
               
                   
                 259 
                 F11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 564 
                 591 
                 0.19 
                 86,499 
                 27 
               
               
                   
                 260 
                 G2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 567 
                 596 
                 0.01 
                 49,703 
                 29 
               
               
                   
                 261 
                 G3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 582 
                 0.07 
                 68,924 
                 21 
               
               
                   
                 262 
                 G4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 559 
                 582 
                 0.17 
                 124,394 
                 23 
               
               
                   
                 263 
                 G5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 558 
                 579 
                 0.09 
                 45,034 
                 21 
               
               
                   
                 267 
                 G6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 569 
                 609 
                 0.12 
                 54,371 
                 40 
               
               
                   
                 268 
                 G7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 563 
                 590 
                 0.15 
                 71,670 
                 27 
               
               
                   
                 274 
                 G8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 563 
                 585 
                 0.01 
                 60,686 
                 22 
               
               
                   
                 275 
                 G9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 561 
                 585 
                 0.15 
                 65,080 
                 24 
               
               
                   
                 277 
                 G10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 576 
                 0.14 
                 79,908 
                 20 
               
               
                   
                 281 
                 G11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 557 
                 577 
                 0.12 
                 52,723 
                 20 
               
               
                   
                 282 
                 H2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 556 
                 574 
                 0.05 
                 27,460 
                 18 
               
               
                   
                 283 
                 H3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 595 
                 637 
                 0.04 
                 31,579 
                 42 
               
               
                   
                 284 
                 H4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 544 
                 571 
                 0.12 
                 44,211 
                 27 
               
               
                   
                 285 
                 H5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 560 
                 586 
                 0.16 
                 60,686 
                 26 
               
               
                   
                 289 
                 H6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2- 
                 557 
                 577 
                 0.14 
                 65,629 
                 20 
               
               
                   
                 290 
                 H7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA2-  
                 559 
                 574 
                 0.01 
                 19,771 
                 15 
               
               
                   
                 292 
                 H8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 560 
                 581 
                 0.14 
                 63,158 
                 21 
               
               
                   
                 294 
                 H9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 558  
                 581 
                 0.14 
                 67,277 
                 23 
               
               
                   
                 298 
                 H10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA2- 
                 556 
                 575 
                 0.09 
                 49,703 
                 19 
               
               
                   
                 299 
                 H11 
                   
                   
                   
                   
                   
               
               
                 BDNCA- 
                 BDNCA-  
                 BDNCA3- 
                 563  
                 590 
                 0.21 
                 102,151 
                 27 
               
               
                 III 
                 300 
                 A2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA3- 
                 567  
                 603  
                 0.24 
                 85,950  
                 36 
               
               
                   
                 301 
                 A3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 553  
                 576 
                 0.15 
                 43,112  
                 23 
               
               
                   
                 302 
                 A4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556  
                 575 
                 0.12 
                 70,847 
                 19 
               
               
                   
                 303 
                 A5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 561  
                 586 
                 0.02  
                 18,673 
                 25 
               
               
                   
                 304 
                 A6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 568  
                 606 
                 0.22 
                 153,227 
                 38 
               
               
                   
                 305 
                 A7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 561  
                 595 
                 0.01 
                 22,792 
                 34 
               
               
                   
                 307 
                 A8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 555  
                 575  
                 0.10  
                 63,432 
                 20 
               
               
                   
                 308 
                 A9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 566  
                 613 
                 0.11 
                 38,444  
                 47 
               
               
                   
                 310 
                 A10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 555  
                 575 
                 0.09 
                 57,117 
                 20 
               
               
                   
                 311 
                 A11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 554  
                 575  
                 0.11 
                 52,998 
                 21 
               
               
                   
                 314 
                 B3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 655 
                 weak 
                 weak 
                 15,652 
                   
               
               
                   
                 315 
                 B4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 575  
                 621 
                 0.09 
                 32,677 
                 46 
               
               
                   
                 316 
                 B5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 557  
                 579 
                 0.08  
                 45,858 
                 22 
               
               
                   
                 317 
                 B6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 564  
                 589 
                 0.02 
                 18,398 
                 25 
               
               
                   
                 318 
                 B7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 598  
                 656 
                 0.05 
                 54,096 
                 58 
               
               
                   
                 319 
                 B8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 557 
                 581 
                 0.10 
                 59,588 
                 24 
               
               
                   
                 320 
                 B9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 561 
                 588  
                 0.14 
                 67,826 
                 27 
               
               
                   
                 322 
                 B10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 578  
                 0.10 
                 60,137 
                 22 
               
               
                   
                 323 
                 B11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 558 
                 581  
                 0.13  
                 52,723 
                 23 
               
               
                   
                 325 
                 C2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 576  
                 0.08 
                 40,915 
                 20 
               
               
                   
                 326 
                 C3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 557 
                 576  
                 0.10 
                 48,330 
                 19 
               
               
                   
                 331 
                 C4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 575  
                 0.05  
                 27,735 
                 19 
               
               
                   
                 332 
                 C5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 560 
                 586  
                 0.12 
                 63,707  
                 26 
               
               
                   
                 333 
                 C6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 569 
                 602  
                 0.08 
                 41,739 
                 33 
               
               
                   
                 335 
                 C7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 562 
                 585  
                 0.12 
                 63,982 
                 23 
               
               
                   
                 336 
                 C8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 532 
                 549  
                 0.04 
                 36,247  
                 17 
               
               
                   
                 340 
                 C9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 558 
                 580  
                 0.10 
                 58,764 
                 22 
               
               
                   
                 341 
                 C10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 562 
                 588  
                 0.06 
                 28,284  
                 26 
               
               
                   
                 343 
                 C11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 621 
                 weak 
                 weak 
                 12,082 
                   
               
               
                   
                 345 
                 D2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-  
                 BDNCA3-  
                 561 
                 584  
                 0.02 
                 16,476 
                 23 
               
               
                   
                 346 
                 D3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 569 
                 604  
                 0.08 
                 34,874 
                 35 
               
               
                   
                 347 
                 D4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 578  
                 0.03  
                 36,247 
                 22 
               
               
                   
                 349 
                 D5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 560 
                 581  
                 0.07 
                 34,874 
                 21 
               
               
                   
                 350 
                 D6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 570 
                 614  
                 0.01 
                 15,652 
                 44 
               
               
                   
                 353 
                 D7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 564 
                 588  
                 0.04 
                 26,636 
                 24 
               
               
                   
                 359 
                 D8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 558 
                 576  
                 0.08 
                 41,190 
                 18 
               
               
                   
                 363 
                 D9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 553 
                 569  
                 0.07  
                 45,584 
                 16 
               
               
                   
                 365 
                 D10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 579 
                 629  
                 0.02 
                 38,444 
                 50 
               
               
                   
                 366 
                 D11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 578 
                 601  
                 0.03 
                 21,968 
                 23 
               
               
                   
                 368 
                 E2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 507 
                 585 
                 0.08 
                 34,050 
                 78 
               
               
                   
                 370 
                 E3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 596 
                 647 
                 0.03 
                 29,931 
                 51 
               
               
                   
                 371 
                 E4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 565 
                 594 
                 0.13 
                 69,474 
                 29 
               
               
                   
                 372 
                 E5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 591 
                 654 
                 0.02 
                 21,968 
                 63 
               
               
                   
                 375 
                 E6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 552 
                 575 
                 0.09 
                 84,027 
                 23 
               
               
                   
                 376 
                 E7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 590 
                 weak 
                 weak 
                 58,215 
                   
               
               
                   
                 377 
                 E8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 566 
                 597 
                 0.16 
                 92,540 
                 31 
               
               
                   
                 378 
                 E9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 555 
                 578 
                 0.11 
                 60,686 
                 23 
               
               
                   
                 384 
                 E10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 555 
                 573 
                 0.09 
                 53,822 
                 18 
               
               
                   
                 392 
                 E11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 591 
                 629 
                 0.04 
                 26,636 
                 38 
               
               
                   
                 393 
                 F2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 507 
                 531 
                 0.01 
                 16,476 
                 24 
               
               
                   
                 396 
                 F3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 573 
                 613 
                 0.01 
                 38,444 
                 40 
               
               
                   
                 397 
                 F4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 569 
                 604 
                 0.11 
                 39,817 
                 35 
               
               
                   
                 401 
                 F5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 564 
                 598 
                 0.08 
                 43,661 
                 34 
               
               
                   
                 403 
                 F6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 574 
                 0.03 
                 18,124 
                 18 
               
               
                   
                 404 
                 F7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 558 
                 582 
                 0.04 
                 19,771 
                 24 
               
               
                   
                 405 
                 F8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 555 
                 575 
                 0.07 
                 42,288 
                 20 
               
               
                   
                 407 
                 F9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 557 
                 577 
                 0.14 
                 87,597 
                 20 
               
               
                   
                 409 
                 F10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 524 
                 547 
                 0.09 
                 49,428 
                 23 
               
               
                   
                 414 
                 F11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 519 
                 weak 
                 weak 
                 13,730 
                   
               
               
                   
                 417 
                 G3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 526 
                 weak 
                 weak 
                 24,439 
                   
               
               
                   
                 421 
                 G4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 569 
                 594 
                 0.10 
                 61,510 
                 25 
               
               
                   
                 423 
                 G5 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 562 
                 583 
                 0.11 
                 46,957 
                 21 
               
               
                   
                 425 
                 G6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 602 
                 630 
                 0.03 
                 43,112 
                 28 
               
               
                   
                 427 
                 G7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 506 
                 657 
                 0.02 
                 17,025 
                 151 
               
               
                   
                 428 
                 G8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 566 
                 599 
                 0.14 
                 64,531 
                 33 
               
               
                   
                 429 
                 G9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 567 
                 601 
                 0.09 
                 37,895 
                 34 
               
               
                   
                 430 
                 G10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 554 
                 573 
                 0.04 
                 19,497 
                 19 
               
               
                   
                 431 
                 G11 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 564 
                 587 
                 0.11 
                 43,387 
                 23 
               
               
                   
                 433 
                 H2 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 576 
                 0.13 
                 61,510 
                 20 
               
               
                   
                 435 
                 H3 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 556 
                 575 
                 0.09 
                 47,506 
                 19 
               
               
                   
                 437 
                 H4 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 565 
                 602 
                 0.17 
                 43,661 
                 37 
               
               
                   
                 440 
                 H6 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 564 
                 587 
                 0.04 
                 23,341 
                 23 
               
               
                   
                 109 
                 H7 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 566 
                 581 
                 0.02 
                 32,952 
                 15 
               
               
                   
                 210 
                 H8 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 565 
                 594 
                 0.13 
                 47,780 
                 29 
               
               
                   
                 41 
                 H9 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA- 
                 BDNCA3- 
                 569 
                 608 
                 0.06 
                 25,538 
                 39 
               
               
                   
                 458 
                 H10 
                   
                   
                   
                   
                   
               
               
                   
                 BDNCA-0 
                 BDNCA3- 
                 495 
                 530 
                 0.06 
                 40,641 
                 35 
               
               
                   
                   
                 H11 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Pancreatic Islet Imaging Probe Development 
     Pancreatic islets imaging is an emerging area in diabetes research, with most published studies describing either in vitro or ex vivo attempts to visualize pancreatic cells using various imaging techniques 1, 2 . Imaging techniques that accurately distinguish viable pancreatic islets enable better understanding of the critical factors involved in diabetic research in both clinical and experimental medicine 3, 4 . To conventionally identify pancreatic islets, it is necessary to perform immunostaining on pancreas sections directly, to induce transgenic mice expressing reporter genes 5  or to introduce bioluminescent genes 6, 7  linked to their promoters. Such methods mostly serve as corroborative optical imaging tools and they are also not applicable for direct islets staining for further advancement of diabetes diagnosis research. Optical imaging techniques using fluorescence have several advantages such as detectability, efficiency and applicability in bioimaging probe development. Hence, using combinatorial chemistry, we have in the past developed optical imaging probes based on the Diversity Oriented Fluorescence Library (DOFL) composed of thousands of intrinsically fluorescent small molecules and successfully applied them to detect specific biomolecules 8  or cells (muscle cell 9  and pluripotent stem cells 10 ). In addition, we also discovered the glucagon imaging probe, GY (Glucagon Yellow) which can stain live alpha cells 11 . 
     To discover pancreatic islet selective probes, we expanded the other two types of BODIPY library generated from the original GY (Glucagon Yellow) structure. We started with high throughput cell-based screening using mouse acinar cells composed of exocrine tissue, mouse Alpha TC-1 cells and Beta TC-6 cells composed of endocrine pancreas islets. Those three different types of cells were prepared side by side in 384-well plates and incubated with 1 μM of BODIPY compounds for different incubation times ranging from 1 hr to 48 hours. The fluorescence images were acquired on an automated imaging microscope system ImageXpress Micro™. The fluorescence intensity of the stained cells was analyzed using MetaXpress® image processing software. Based on the calculated intensity and manual confirmation, we selected 3 prospective compounds (BDNCA5, BDNCA16 and BDNCA325) that stained either both alpha cells and beta cells or beta cells only as compared with acinar cells. Surprisingly, only one compound, BDNCA-325 ( FIG. 1 a   ) showed selectivity in staining the beta TC-6 cells ( FIG. 1 b   ). 
     Next, we further evaluated the 3 compounds in vitro by adding the compounds on cryo-pancreatic sections. It was performed by washing the pancreas sections with PBS before incubating the stained sections with 1 μM of compound for one day at room temperature. The next day, the sections were washed with PBS buffer to remove nonspecific staining. All the three compounds stained the pancreatic islets specifically and the staining was also confirmed by immunostaining with an insulin antibody. The promising fact that these compounds were able to detect pancreatic islets in vitro, suggested the possibility that they can be applied in vivo. 
     Based on this, we injected the compounds intravenously into the mouse to determine if the staining pattern was similar to that observed in vitro. Each of the 3 compounds was administrated into the mouse separately for one hour before dissection. The cryo-pancreas sections were washed once in PBS before observation. Unlike the previously examined results, the compounds stained the whole pancreas tissue nonspecifically (Data not shown). 
     To develop selective pancreatic islet staining compounds for in vivo conditions, we synthesized several derivatives of the original beta cell selective compound BDNCA325 by changing the reactive groups. The modified compound which was named PiY was synthesized, removing the alpha-chloro part which can bring out nonspecific staining by covalent binding in vivo ( FIG. 2 a   ). This PiY compound was tested in vivo to determine if it shows selectivity to pancreatic islets. The optimum time required for the staining of the islets was also investigated in a time dependent manner by injecting the compound at different time points, from 0.5 hr to 4 hrs. It was clearly monitored that the dye slowly starts to invade the exocrine at around two hours to four hours incubation. 0.5 hour to 1 hour incubation was also sufficient to allow visualization of stained islets without any nonspecific staining ( FIG. 2 a   ) Moreover, it was possible to observe the islets at low magnification using the fluorescent microscope after one hour incubation with the compound ( FIG. 2 b   ). Immunostaining revealed co-localization of BDNAC325 stained islets with both insulin and glucagon antibodies ( FIG. 2 c   ). 
     Eventually these findings resulted in the discovery of the pancreatic islet probe known as “PiY(BDNAC325)”, (Pancreatic islet Yellow) (λex/λem=558/585 nm) Extinction coefficient=27700; Quantum yield=0.05) which distinguished pancreatic islets from surrounding exocrine tissues in vivo conditions. To compare PiY with other compounds, we investigated the Newport Green™ DCF diacetate 12 , a commercially available fluorescent compound for human pancreatic islets, to see if it stains mouse pancreatic islets in vivo. This reference compound had no selectivity for mouse pancreatic islet after injection, although the Newport Green™ DCF diacetate could moderately stain pancreatic islet in vitro. PiY staining as detected in vivo can serve as a platform to improve conventionally available imaging techniques as mentioned previously. 
     To induce the PiY into mice, it was dissolved in 1% of PEG-4600 and 0.1% of tween-20 from 1 mM of DMSO stock solution. Thus, we considered the effect of both PiY and the solvent on insulin secretion. The stained islets were isolated from pancreas after one hour administration and followed on culture condition. It was noted that both PiY and solvent as described above interfered in neither basal nor secretary insulin regardless of glucose stimulation ( FIG. 2 d   ). Therefore PiY can be used as imaging tool for pancreas islets in vivo without hampering the functional effect of insulin secretions involved in diabetic disease. 
     For further application, we employed PiY into streptozotocin (STZ) induced type 1 diabetic mouse model to observe healthy pancreatic islets staining vs. diabetic islets. First of all, we pretested to verify whether PiY is able to affect blood glucose level change, not streptozotocin. As expected, there were no detectable changes, for at least a month, for either blood glucose level or body weight at high dosage (up to 150 μM). Using the STZ model of type 1 diabetes, we observed that unhealthy islets from STZ induced mouse had only a few PiY stained islet composed cells ( FIG. 3 a   ). To confirm PiY staining, pancreas sections were immunostained with insulin antibody and the obtained images correlated with PiY staining ( FIG. 3 b   ). Moreover, we managed to statistically analyze the stained islets by measuring sum intensity and by counting the (islet) number. To start with, we set up 3 groups of STZ and control mice respectively depending on blood glucose level of STZ mouse before PiY injection, to collect the statistical data. The glucose level of STZ mouse of group 1 was over 20 mM. For group 2, it was between 15-18 mM and for group 3, it was around 12-15 mM. The glucose level of control mice were detected more or less 10 mM in all three groups. 50 μM of PiY was administrated into mouse tail vain for 1 hour and the section was prepared as described previously. Staining of islets was observed. We were also able to calculate the intensity in control vs. diabetic model by annotation and measurement program which can detect fluorescence of specified area automatically. The intensity of healthy islets was perceived over two times brighter than diabetic islets in all three groups. In addition, a total number of healthy islets were counted higher than STZ induced diabetic mice. Therefore PiY enables differentiation of healthy islet vs. diabetic islet by staining healthy islets specifically. 
     Example 3 
     Microglia Cells Imaging Probe 
     Microglia cells are immune cells of the brain that are responsible for day-to-day surveillance and maintenance of neural function. They are derived from hematopoietic precursors during early embryogenesis and constitute approximately 5-10% of the neural cell population. 13  Microglia cells are distributed ubiquitously throughout the brain and are characterized by a ramified cellular phenotype in the absence of stimulation. 14  These cells respond dynamically to cellular damage or invading pathogen stimulus by proliferation and migration towards the afflicted regions. This may occur in response to chemokine stimulus or through pattern recognition receptors such as Toll-like receptor 4 (TLR4) which recognize pathogen components such as bacterial LPS. 15  On the recognizing these signals, the microglia secrete potent and potentially cytotoxic cytokines such as interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). These cytokines play a role in activating a downstream inflammatory cascade, which in a healthy immune system facilitates the clearance of potential pathogens. However, excessive microglia activity has also been associated the pathogenesis of neuroinflammatory diseases. 16  As a result, microglia localization and activity within the brain are of significant interest to those wishing to study their general function and involvement in disease. 
     The cellular organization of the brain is complex and its tight cellular junctions form the infamous blood-brain-barrier (BBB) thus making drug access possible only for select small molecules. Microglia cells, in particular, are challenging subjects to study due to their relatively low numbers in the brain (5-10%) and slow growth in vitro (3). As a result, primary cultures of microglia are often tedious to prepare to sufficient purity and scale and have a low yield. 17  Current methods of labeling microglia in an in vitro setting are predominantly antibody mediated—with the cell surface markers Iba1, CD11b and CX3CR3 being some of the more common targets. This usually involves the expensive and time-consuming process of acquiring and labeling the cells with the relevant antibodies. More recently, conjugation of the above markers to fluorescent proteins such as EGFP has become popular for live cell imaging purposes. However, the process of generating these transgenic animals is tedious and lengthy thereby making the labeling of these cells by this means a challenging process. Thus the development of new easy-to-use fluorescent probes capable of directly labeling live cells would be an invaluable tool for microglia tracking and in vitro and in vivo imaging purposes. A significant number of microglia-targeted probes have been developed to work in an in vivo setting. Most of these probes are ligands of the 18 kDa translocator protein, also known as the peripheral benzodiazepine receptor (PBR). 18  An example is the popular PET probe, 11C-(R)-PK11195 that is a PBR selective ligand. PBR has been found to be expressed on the outer mitochondrial membrane of activated microglia and the signal derived from the administration of the 11C-(R)-PK 11195 probe is generally interpreted to be indicative of the presence of inflammation and neurodegeneration in the brain. 18  However, the use of such radioligand probes has been plagued with problems such as a low target to background ratio and limited target delivery. 19  There has also been some debate as to what extent PK11195 signal can be attributed to activated microglia. As such, there is still a continued drive for the development of more microglia specific probes to extend the scope of in vitro and in vivo studies. 
     Chemical biology is an emerging new field that applies the diversity of chemical structures for targeted biological applications. 20  The objective of our study was to identify neural cell type specific fluorescent probes from our Diversity Oriented Fluorescence Library (DOFL) for application in cellular imaging systems. In this case, we identified a fluorescent probe that is specific for primary microglia from a high throughput in vitro culture screening system. Using fluorescence microscopy and flow cytometry analysis, we were able to demonstrate that these probes were selective for primary microglia in primary neural cell cultures and exhibited similar specificity for microglia-derived cell lines. Cytotoxicity and functional assays also showed that these cells remained viable in the presence of staining and retained their ability to express their relevant cytokines and cellular mediators in response to stimulation. Lastly, the efficacy and utility of our small molecule fluorescent probes may also be demonstrated by their use in numerous cellular images applications such as cellular detection and tracking with the potential for further development as in vivo imaging probes. 
     BDNCA164 (BDNCA1 H5) Exhibit Specificity for Primary Microglia Cells. 
     Stained cells were subsequently immunostained for Iba1, a known surface marker for microglia to confirm for cell type specificity ( FIG. 4 ). Microglia cells in the brain are also known to constitutively express the chemokine receptor, CX3CR1 that is a cell surface marker. To test for specificity and further rule out that the compounds may be staining other neural cell types, 500 nM of compound was applied to cultures of mixed primary neural cell cultures derived from CX3CR1-GFP transgenic mice and checked for GFP and compound co-localization. 
     Our compounds appear to localize within the cytoplasm of the microglia excluding the nucleus, appearing as bright circular dots with a dark centre. To further confirm the specificity of our compounds for microglia in heterogeneous neural cell systems, we used flow cytometry analysis to confirm that our compound signal coincides with GFP positive microglia in mixtures of primary neural cells derived from CX3CR1-GFP transgenic mice ( FIG. 5 ). The signal from GFP-Texas Red double positive microglia cells increased in a dose dependent manner (data not shown) although higher concentrations gave rise to more non-specific staining depending on the incubation duration. To prove that primary microglia were still distinguishable from primary neural cells in the absence of the CX3CR1-GFP marker, neural cells derived from normal FVB/N mice were also stained and analyzed via flow cytometry ( FIG. 6 ). 
     Compound Specificity is Conserved in the BV2 Microglia Cell Line. 
     Our fluorescent compounds were developed using primary neural cells based on the premise that primary cells would reflect the cells in their true native state and represent a reliable benchmark for specificity testing. Cell lines, while highly prolific have been known to spontaneously incur genomic or functional changes that may not reflect their true state. 21  Thus given the specificity of our compounds for primary microglia, we were interested to see if this was also conserved in primary microglia-derived cell lines. To demonstrate this, we used the primary microglia-derived retrovirus transformed cell line, BV-2 in co-culture with mouse embryonic fibroblasts. Compound stained BV-2 cells were clearly distinguishable in co-culture with MEF and showed a similar staining pattern (largely cytoplasmic) with the primary derived microglia thus demonstrating that the specificity of compound towards primary microglia is conserved in primary microglia-derived cell lines. Compound stained microglia retain viability and functional response 
     A central concern in the use of live cell probes is their potential to interfere with normal cellular function, which would be undesirable for downstream applications. Using the MTS assay at a 24 h time point, we can show that both probes are not cytotoxic at their concentrations and durations used (500 nM, 1 hour) and concentrations above this induce only minimal cytotoxicity that are unlikely to affect the cells at our given time points ( FIG. 7A ). Cell proliferation studies also showed that cells retain their ability to proliferate in vitro in the presence of the compounds with no detrimental effect ( FIG. 7B ). 
     The ability of microglia to release nitric oxide in response to stimulation is an important immune defense, which facilitates the removal of potentially harmful pathogens. Here, we show using the Griess assay for nitrate detection that compound stained microglia retain their ability to produce nitric oxide in response to lipopolysaccharide (LPS) and IFN-γ stimulus and that these levels of nitric oxide production are comparable to LPS or IFN-γ stimulation alone. The incubation of microglia with the compounds alone also does not induce significant nitric oxide release as compared to the LPS stimulated controls thus emphasizing the benign effects of the probes on microglia function. This facilitates their use in labeling processes that may require a functional response further downstream. 
     Cellular Labeling does not Significantly Affect Cytokine Expression. 
     In addition to nitric oxide release, the cytokine cascade is another important mechanism for counteracting pathogen attack. Bacteria-derived cellular components such as LPS and IFN-γ are known microglia activating agents that induce microglia expression and secretion of pro-inflammatory cytokines such as IL-6 and TNF-α by the activation of Toll-like receptor 4 (TLR4) signaling molecules on the cell surface. 15  Labeling of microglia cells with our fluorescent probes followed by LPS activation and measurement of cytokine expression by real time PCR showed that cytokine expression in response to LPS is not significantly impaired by compound staining ( FIG. 8A ). Similarly, incubation with the compounds alone did not induce any significant increase in cytokine expression thus indicating that the compounds do not display any activating effects ( FIG. 8B ). 
     Compounds were accessed for potential activating effects on the microglia by measuring nitric oxide production. BV2 cells were pretreated for 1 h with each compound (0.5-5 μM). LPS treated BV2 cells were used as a positive control. After incubation for 24 h, nitric oxide production was measured using the Griess assay and concentrations determined by comparison to a standard curve. 
     Applications for Microglia-Specific Probes. 
     Cellular Detection and Tracking. 
     The role of microglia as immunological ‘sentinels’ in the brain and their ability to mediate the dual functions of neuroprotection and neuroinflammation make them popular candidates as tools for cellular tracking and detection in vitro or in vivo settings. As highlighted by Saura 2007 22 , the presence of contaminating microglia in astroglial cultures can skew experimental results significantly. BDNCA 164 can be applied reversibly as a quick and easy method of accessing the amount of microglia contamination in a culture. Alternatively, as shown in fluorescence activated cell sorting (FACS) may be used to remove contaminating populations of microglia or purify them for subsequent reculture. 
     BDNCA 164 staining does not succumb to methanol washing and is effective in both serum-containing and serum-free conditions thus making ideal for extended duration cellular tracking. To demonstrate this, we stained primary mixed glial cells derived from CX3CR1-GFP mice with BDNCA 164 and imaged them over a time course of 24 hours. The fluorescent signal from our compound makes it possible to trace the movement of primary microglia cells in a culture with no significant phototoxicity or photobleaching. Depending on the experimental design, this compound can thus be used can be used for more complex tracking of microglia in vitro, such as the detection of their chemoattraction towards a given stimulus. 
     In vivo Studies and Inflammation Detection. 
     As demonstrated above, our compounds have mainly been optimized to work in vitro conditions. However it can potentially be applied in vivo conditions as well. Its efficacy both in vitro and in vivo facilitates its further development as an in vivo imaging probe for a wide range of applications. This may be carried out through the incorporation of radioactive isotopes such as  18 F,  11 C into the compound structure thus making it suitable for detection by PET analysis. Neural systems aside, our probes may also possess some affinity for macrophages in the blood, which derive from a similar hematopoietic lineage as brain microglia (data not shown). This may also be another potential area for probe development for inflammation detection and therapeutic delivery in the future. 23    
     Example 4 
     BDNCA1 H5 is Localized Predominantly in the Mitochondria of Microglia Cells 
     High magnification imaging and co-staining with MitoTracker Green (Invitrogen) shows that a majority of the dye staining is localized in the mitochondria of the cells with some background staining in the cytosol ( FIG. 10 a   ). The dye and MitoTracker Green showed good co-staining (Pearson&#39;s coefficient=0.93) whereas co-staining with other cell organelle markers (Lysotracker, showed poor co-staining (Pearson&#39;s coefficients of 0.47) ( FIG. 10 b   ). This strongly suggests that the dye is binding to a target protein present in cell mitochondria. 
     To further elucidate this, the mitochondrial and cytosolic extracts of BV2 stained cells were isolated and run on SDS-PAGE. Proteins bound by the dye are visible as fluorescent bands under the appropriate excitation and emission wavelengths ( FIG. 10 c   ). For BV2 cells, this was visible as a distinct bright band at the 25 kD region that was present in the mitochondrial and not in the cytosol fraction as confirmed by western blotting for COX IV (mitochondrial loading control). This provides further evidence that our target protein is highly expressed in the mitochondria. 
     Activated Microglia Exhibit Brighter Compound Staining. 
     We were interested to see if BDNCA 164 (BDNCA1 H5) could also serve as a reporter of cell activation status by discriminating resting and activated microglia by measurement of fluorescence intensity. To investigate this, BV2 cells activated by LPS were stained with BDNCA1 H5 and analyzed in parallel with non-activated cells. Both microscope image and flow cytometry data showed that activated microglia are up to 2 times brighter than their non-activated counterparts ( FIG. 11 a  and 11 b   ). 
     Material and Methods 
     Chemical Synthesis 
     All reactions were performed in oven-dried glassware under a positive pressure of nitrogen. Unless otherwise noted, starting materials and solvents were purchased from Aldrich and Acros organics and used without further purification. Analytical TLC was carried out on Merck 60 F254 silica gel plate (0.25 mm layer thickness) and visualization was done with UV light. Column chromatography was performed on Merck 60 silica gel (230-400 mesh). NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer. Chemical shifts are reported as 6 in units of parts per million (ppm) and coupling constants are reported as a δ value in Hertz (Hz). Mass of all the compounds was determined by LC-MS of Agilent Technologies with an electrospray ionization source. Spectroscopic measurements were performed on a fluorometer and UV/VIS instrument, Synergy 4 of bioteck company and Gemini XS fluorescence plate reader. The slit width was 1 nm for both excitation and emission. Relative quantum efficiencies were obtained by comparing the areas under the corrected emission spectrum. The following equation was used to calculate quantum yield
 
Φ x=Φst ( I   x   /I   st )( A   st   /A   x )(η x   2 /η st   2 )
 
where Φst is the reported quantum yield of the standard, I is the integrated emission spectrum, A is the absorbance at the excitation wavelength, and ηx is the refractive index of the solvents used. The subscript x denotes unknown and st denotes standard. Rhodamine B was used as standard.
 
Cell Culture
 
     Primary neural cultures were generated from the whole brains of P1-3 FVB/N mice by trypsinization and trituration methods in accordance with the animal handling regulations of our institution. Briefly, fresh brains were incubated with 0.25% trypsin for 1 hour at 37° C., followed by neutralization with Fetal Bovine Serum (FBS). A single cell suspension was generated by sequential trituration using pipette tips of decreasing diameter. Finally, the cell suspension was filtered using a 40-micron strainer. Cells were grown in DMEM/F12 (Gibco) with 10% FBS and 1% penicillin-streptomycin (Gibco). BV2 and MEF cells were cultured in DMEM/F12+2% FBS and DMEM+20% FBS respectively with 1% penicillin streptomycin. 
     In vitro PiY Staining and Immunohistochemistry 
     The pancreas was embedded in optimal cutting temperature compound after 4% PFA fixation for 3 hr and was sectioned into 10 μm thick slices. The sections on slides were air-dried at room temperature for 30 min and stored at −20° C. until use. The sections were washed with cold PBS for 5 min gently. 1 uM of PiY was directly applied on sections for over night at room temperature. Next day, the nonspecific staining was removed using cold PBS three times for 10 min and blocked with 2% bovine serum albumin for 1 hr before incubation with a guinea pig polyclonal antibody against Insulin (diluted 1:1000, DAKO) and rabbit monoclonal antibody for Glucagon (diluted 1:50, Cell Signaling) overnight at 4° C. The antibody was visualized by a Cy5-conjugated goat anti-guinea pig secondary antibody and Cy5-conjugated goat anti-rabbit secondary antibody, respectively (diluted 1:500, Invitrogen). 
     In vivo Staining of PiY 
     The 1 mM of DMSO stock was dissolved in PBS with 1% of 4600PEG and 0.1% of tween20 to administrate 50 uM of PiY solution into mouse tail vain for one hour. The pancreas sections were washed with PBS for 5 min after dissection. The sections were observed under fluorescent microscopy on TRITC channel and immunostained with insulin antibody as above. 
     Streptozotocin Administration for Type 1 Diabetic Mouse Model 
     The streptozotocin was dissolved in Na-Citrate buffer solution immediately prior to injection and administrated into intraperitoneal using 150 mg/kg as a final dosage. An experimental set was made up of three mice for control and STZ, respectively. The blood glucose level was checked every 3 days using blood glucose meter (Lifescan Inc., Milpitas, Calif., USA). Three STZ mice were separated into each group depending on blood glucose level such as group 1 (&gt;20 mmol/l), group 2 (15-18 mmol/l) and group 3 (12-15 mmol/l) including each control mouse. Subsequently 50 uM of PiY was injected into control and STZ mice tail vain and kept alive for 1 hr. 
     After that stained islets were observed and carried out the immunostaining using insulin antibody as described. 
     Insulin Secretion Assay 
     Islets were isolated after injection of 50 uM of PiY for 1 hr and cultured in basal medium: Krebs-Ringer-Hepes (KRH) medium (containing, mM: 130 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and 2.56 CaCl2, 1 mg ml-1 BSA, 20 mM Hepes, pH 7.4) supplemented with 3 mM glucose for 2 h at 37° C. For static incubation experiments, similar-sized islets from a single mouse (3 islets per tube) were first washed with 3 mM glucose-containing KRH medium, placed in eppendorf tube. Basal medium (100 ul) was added and islets were incubated 15 minutes at 37° C. They were then stimulated with 600 ul of 20 mM glucose-containing KRH medium for 15 min after which the incubation medium was collected after 15 min and replaced with fresh 20 mM glucose KRH. Islets were incubated further 15 min and the medium was collected. Insulin concentration was measured using a Mouse insulin ELISA kit (Mercodia). This experiment was conducted as duplicate with each two mice. 
     Statistical Islet Analysis by PiY 
     The three serial sections were prepared on slide numbered Slide No 1. Next sections have over 100 μm size gap to avid the same islets overlapped slide by slide. A total 300 sections per mouse were observed to analyze islets intensity and islets number. By using annotation and measurement software, the intensity of stained islets were automatically detected and calculated at the same time. After automatic analysis, the islets number was counted by manually. The data were normalized by STZ value. 
     High Throughput Screening 
     Primary neurons and primary astrocyte enriched cultures were generated using the adherence isolation protocol by Jana et al 32 . Primary microglia enriched cultures were generated by adherence methods and harvested using the mild trypsinization method as described by Saura et al 17 . Neuron, astrocyte and microglia enriched cultures were plated on to 384 well plates and incubated with 500 nM of fluorescent compound for 1-2 hours before screening using the ImageXpress high throughput system. Images were scored using intensity and image-based analysis for the identification of hit compounds. 
     Immunocytochemistry 
     Hit compounds from screening were subsequently tested in cultures of mixed primary neural cells (i.e. whole brain cultures) for specificity. Due to the phenotypic heterogeneity observed in neural cells, it is necessary to confirm cell type by immunostaining. As such, ‘compound bright’ cells in mixed cultures were imaged by fluorescence microscopy using the ECLIPSE Ti microscope (Nikon Instruments Inc) on the NIS Elements 3.10 software. Cells were then fixed in 4% paraformaldehyde (Sigma) and immunostained for Iba (Wako). The same cells were then and re-imaged for Iba. 
     Fluorescence Imaging 
     CX3CR1-GFP transgenic mouse pups were a kind gift from Dr. Fluorent Ginhoux. Primary neural cultures from these mice were generated as described above. Cells were stained with 500 nM of compound for 1 hour followed by 1 hour of washing in fresh media before image acquisition. Confocal images were generated on an AIR confocal microscope (Nikon Instruments Inc.) using NIS Elements 3.10 software at 100× magnification. 
     Flow Cytometry 
     Primary neural cultures were generated from the brains of CX3CR1-GFP mice as described above. Cells were stained with 500 nM of compound for 1 hour and washed with PBS before acquisition on the BD LSR II analyzer. Cells were acquired using the appropriate filters for GFP and Texas Red wavelengths. 
     Cytotoxicity Assays 
     Cytoxicity assays were carried out using the MTS reagent kit (Promega) on 24 h compound treated BV-2 microglia cells in accordance with the manufacturer&#39;s instructions. Cell proliferation was measured by counting of Hoescht labeled nuclei (Invitrogen) using the ImageXpress high throughput screening system at Days 0, 1 and 2. 
     Cytokine Expression 
     BV2 microglia cells were pretreated for 1 h with 500 nM of compound before addition of 100 ng/ml of LPS in fresh media. After 6 hours, total RNA was isolated from the cells using the RNeasy Mini Kit (QIAGEN Inc.) according to the manufacturer&#39;s instructions and cytokine expression levels of IL-6 and TNF-α measured by RT-PCR. 
     Gene Expression for the Cytokines IL-6. TNFα and GAPDH was measured using the Power SYBR® Green RNA-to-CT™ 1-Step Kit (Applied Biosystem) on a StepOne™ Real-Time PCR System (Applied Biosystems). mRNA levels of the above mentioned genes were normalized to GAPDH expression levels using the Q-gene relative expression software tool.
 
NCBI References for the Below Sequences:
       Mus musculus  glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNA   

     Accession: NM_008084.2
       Mus musculus  tumor necrosis factor (Tnf), mRNA   

     Accession: NM_013693.2
       Mus musculus  interleukin 6 (116), mRNA   

     Accession: NM_031168.1 
     The following primer sequences (5′ to 3′) were used in this study: 
                            SEQ ID. No.: 1                             mGAPDH-F1   AAGGGCTCATGACCACAGTC                                     SEQ ID. No.: 2                             mGAPDH-R1   GGATGCAGGGATGATGTTCT                                     SEQ ID. No.: 3                             mTNFα-F1   TAGCCAGGAGGGAGAACAGA                                     SEQ ID. No.: 4                             mTNFα-R1   TTTTCTGGAGGGAGATGTGG                                     SEQ ID. No.: 5                             mIL-6-F1   CCGGAGAGGAGACTTCACAG                                     SEQ ID. No.: 6                             mIL-6-R1   TCCACGATTTCCCAGAGAAC            
Nitric Oxide Production
 
     BV2 cells were pretreated for 1 hour with 500 nM of compound before addition of 100 ng/nl of LPS or 20 U/ml (20 ng/ml) of IFN-γ in fresh media. After incubation for 24 hours, 50 μl of cell culture media was mixed with an equal volume of Griess reagent (Sigma) in a 96 well plate. Light absorbance was measured at 540 nm. Nitric oxide concentration was determined by comparison with a standard curve generated from a titration of sodium nitrite. 
     Binding and Washing 
     To assess mode of binding, BV2 microglia were stained with 500 nM of compound in serum free media for one hour. Cells were subsequently washed for 1 hour with PBS, fresh serum free media, serum containing media (10%) or methanol. They were then imaged by fluorescence microscopy using the ECLIPSE Ti microscope (Nikon Instruments Inc) on the NIS Elements 3.10 software. 
     Cell Tracking 
     Cell tracking was carried out on mixed glial cell cultures stained for 1 h with 500 nM of BDNCA1 H5. This was followed by 1 h of washing in serum containing media. Cell media was replaced once more before video acquisition. For cell tracking, videos were acquired on the Biostation IM (Nikon) at 100× magnification for a duration of 24 hours. 
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     The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. 
     While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.