Patent Publication Number: US-2018031478-A1

Title: Surface evaluation system using voltage sensitive chromophore

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     Reference is made to commonly assigned, co-pending U.S. patent application Ser. No. ______ (Docket K002020), entitled: “Method for measuring solid-liquid interfacial potentials,” by A. Wexler et al.; to commonly assigned, co-pending U.S. patent application Ser. No. ______ (Docket K002101), entitled: “Method for characterizing a liquid,” by A. Wexler et al.; and to commonly assigned, co-pending U.S. patent application Ser. No. ______ (Docket K002102), entitled: “Method for determining a characteristics difference between liquids,” by A. Wexler et al., each of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to a system for evaluating surface characteristics by measuring a fluorescence emission spectrum of a voltage sensitive chromophore affixed to the surface. 
     BACKGROUND OF THE INVENTION 
     Voltage sensitive dyes (VSD), also known as potentiometric dyes or voltage sensitive chromophores, have been used to determine membrane potentials for biological cells. Such dyes have been designed to become embedded within a cell membrane orthogonal to the membrane surface where they are then exposed to the membrane electric field. U.S. Patent Application Publication 2010/0074847 to Madden et al., describes the use of chromophore probes for optical imaging of biological materials. The use of voltage sensitive dyes, is also discussed by L. Loew in an article titled “Potentiometric dyes: Imaging electrical activity in cell membranes” (Pure &amp; Appl. Chem., Vol. 68, pp. 1405-1409, 1996). The use is very similar to that described U. S. Patent Application Publication 2010/0074847. 
     The absorbance or fluorescence emission of a VSD can be measured for the membrane in both the polarized state and un-polarized state. The shift in the resulting spectrum gives the electrical potential difference between the two states. 
     Two types of VSDs, which vary in their response mechanism, are known. What are considered “slow” VSDs partition within the cell, and their fluorescence intensity is a Nernstian-concentration-dependent response. What are considered “fast” VSD&#39;s do not depend on partitioning and instead respond to the electric field directly via the Stark effect. 
     Experimental strategies for measuring electric field strength (intensity) at charged solid surfaces using a fluorescent dye as a probe particularly in bulk behavior are described by J. Pope et al. in an article entitled “Measurement of electric fields at rough metal surfaces by electrochromism of fluorescent probe molecules embedded in self-assembled monolayers” (J. Am. Chem. Soc., Vol. 114, pp. 10085-10086, 1992), as well as by J. Pope et al. in an article entitled “Measurements of the potential dependence of electric field magnitudes at an electrode using fluorescent probes in a self-assembled monolayer” (J. Electroanalytical Chem., Vol. 498, pp. 75-86, 2001). 
     U.S. Pat. No. 5,156,918 No. (Marks et al.) describes the use of a poly(phenylene ether) to which a pyridine-terminated chromophore is attached and the resulting nonlinear optical material can be covalently attached to solid surfaces as a monolayer for various purposes. 
     Surface potential is a key parameter in colloidal and biological sciences governing interactions between materials such as the attractive or repulsive interaction between materials. Thus, adhesion forces between particles or polymers and a surface that affects particle patterning are directly influenced by surface potentials. Such particles and polymers cover a wide range of materials ranging from biological to inorganic materials. Currently there are two prominent methods to determine surface potentials. However, each of these methods create at least one problem. For example, the method of using streaming potentials described by H. Xie et al. in the article “Zeta potential of ion-conductive membranes by streaming current measurements” (Langmuir, Vol. 27, pp. 4721-4727, 2011) requires expensive equipment and the method is damaging to tested samples. Moreover, the use of atomic force microscopy as described by S. Li et al. in the article “Excluding contact electrification in surface potential measurement using Kelvin probe force microscopy” (ACS Nano, Vol. 10, pp. 2528-2535, 2016) is restricted to very small areas and requires long scanning times. 
     There remains a need for less burdensome and more versatile methods for determining solid-liquid interface electrical field intensities, and for determining the characteristics of liquids. 
     SUMMARY OF THE INVENTION 
     The present invention represents a surface evaluation system for evaluating a surface, including: 
     a chromophore application system for applying a monolayer of a voltage sensitive chromophore to the surface, wherein the applied voltage sensitive chromophore is covalently bound to the surface and has a fluorescence emission spectrum which varies in accordance with a characteristic of the surface; 
     an irradiation source that irradiates the monolayer of the covalently bound voltage sensitive chromophore with actinic radiation; 
     a fluorescence sensing system for measuring a fluorescence emission spectrum from the irradiated monolayer of the covalently bound voltage sensitive chromophore; and 
     an analysis system that analyzes the measured fluorescence emission spectrum to evaluate the characteristic of the surface. 
     This invention has the advantage that characteristics of a surface can be conveniently measured by measuring the fluorescence emission spectrum of a voltage sensitive chromophore attached to the surface. 
     It has the additional advantage that defects and features on the surface that are not detectable with visible radiation can be inspected. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a flowchart of a method for measuring an interfacial electric field intensity according to an exemplary embodiment; 
         FIG. 2A  illustrates an exemplary first reaction useful in the method of  FIG. 1 ; 
         FIG. 2B  illustrates an exemplary second reaction useful in the method of  FIG. 1 ; 
         FIG. 2C  illustrates an exemplary third reaction useful in the method of  FIG. 1 ; 
         FIG. 3A  is a graph showing a Stark shift measured using the method of  FIG. 1  for an exemplary voltage sensitive chromophore with a quartz surface and water; 
         FIG. 3B  is a graph showing a Stark shift measured using the method of  FIG. 1  for an exemplary voltage sensitive chromophore with a quartz surface and methanol; 
         FIG. 3C  is a graph showing a Stark shift measured using the method of  FIG. 1  for an exemplary voltage sensitive chromophore with a quartz surface and acetone; 
         FIG. 4  is a schematic showing a voltage sensitive chromophore bonded to a silica surface; 
         FIG. 5A  is a schematic showing a monolayer of voltage sensitive chromophore where the dipole P is flipped upon excitation with actinic radiation in the presence of an interfacial electric field; 
         FIG. 5B  is an energy diagram illustrating the energy difference resulting from an interfacial electric field; 
         FIG. 6  is a graph illustrating a relationship between the dielectric constant of the liquid and the resulting Stark shift; 
         FIG. 7  is a flowchart of a method for measuring a liquid characteristic according to an exemplary embodiment; 
         FIG. 8  is a flowchart of a method for determining characteristic differences between two liquids according to an exemplary embodiment; 
         FIG. 9  is a graph illustrating a method for comparing two fluorescence emission spectra at first and second wavelengths; and 
         FIG. 10  illustrates an exemplary surface evaluation system for evaluating a surface in accordance with an exemplary embodiment. 
     
    
    
     It is to be understood that the attached drawings are for purposes of illustrating the concepts of the invention and may not be to scale. Identical reference numerals have been used, where possible, to designate identical features that are common to the figures. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The following discussion is directed to various embodiments of the present invention and while some embodiments can be desirable for specific uses, the disclosed embodiments should not be interpreted or otherwise considered to limit the scope of the present invention, as claimed below. In addition, one skilled in the art will understand that the following disclosure has broader application than is explicitly described in the discussion of any embodiment. 
     The invention is inclusive of combinations of the embodiments described herein. References to “a particular embodiment” and the like refer to features that are present in at least one embodiment of the invention. Separate references to “an embodiment” or “particular embodiments” or the like do not necessarily refer to the same embodiment or embodiments; however, such embodiments are not mutually exclusive, unless so indicated or as are readily apparent to one of skill in the art. The use of singular or plural in referring to the “method” or “methods” and the like is not limiting. It should be noted that, unless otherwise explicitly noted or required by context, the word “or” is used in this disclosure in a non-exclusive sense. 
     Each term that is not explicitly defined in the present application is to be understood to have a meaning that is commonly accepted by those skilled in the art. If the construction of a term would render it meaningless or essentially meaningless in its context, the term definition should be taken from a standard dictionary. 
     The use of numerical values in the various ranges specified herein, unless otherwise expressly indicated otherwise, are considered to be approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as the values within the ranges. In addition, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. 
     When a solid is immersed in a fluid, an electric field is generated at the interface. This interfacial electric field can also be characterized by a corresponding interfacial potential difference. In accordance with embodiments of the invention, the interfacial electric field (or the corresponding interfacial potential difference) can be quantified by measuring a wavelength shift of the fluorescence emission spectrum between a fast VSD present in the liquid and a monolayer of the VSD affixed to a solid surface in contact with the same liquid (or via a probe with bound VSD immersed in the liquid). Given the same solvent shell structure and reorganization on excitation for bulk and surface VSD bound at the interface, the fluorescence wavelength shift upon excitation is due to the extra energy needed to flip the chromophore dipole in the interfacial electric field. Chromophore dipole moments for a series of voltage sensitive dyes are available in the literature (for example, see P. Fromherz, “Monopole-dipole model for symmetrical solvatochromism of hemicyanine Dyes,” J. Phys. Chem., Vol. 99, pp. 7188-7192, 1995). The solvatochromic shift of the VSD is cancelled out since both fluorescence spectra (that is, the bound VSD monolayer and dissolved VSD in the liquid) are recorded in the same solvent. 
     In various embodiments, the shift in fluorescence emission spectrum of a bound VSD at a solid/liquid interface can be used to perform various functions such as calculating the electric field strength at the interface between a liquid and a surface, identifying or characterizing liquid, determining characteristic differences between a plurality of liquids, and evaluating surface characteristics/uniformity. 
       FIG. 1  shows a flowchart of a method for determining an interfacial electric field intensity  175  in accordance with an exemplary embodiment. A series of reactions is used to add a monolayer of a voltage sensitive chromophore (i.e., a voltage sensitive dye (VSD)) to a surface  100 . The surface  100  can be a non-biological surface such as a silica surface or a polymer surface. In a first reaction  110 , a linking group  105  including a chromophore component is covalently attached to the surface  100 . The linking group  105  can also be referred to as a “reactive linking molecule.” In an exemplary embodiment, the first reaction  110  is performed by: (a) providing a solution including the linking group; (b) providing a surface capable of reacting with the linking group; and (c) bringing the solution into contact with the surface, thereby reacting the linking group with the surface to attach the linking group to the surface with a covalent linkage. 
       FIG. 2A  illustrates an exemplary first reaction  110  that can be used in accordance with the present invention. In this example, the surface  100  is a silica surface, and the linking group  105  includes a chromophore component  145  and a coupling agent  147 . The coupling agent  147  is adapted to couple the linking group  105  to the surface  100 . The illustrated chromophore component  145  includes a reactive carbocyclic aromatic linking group (in this case a benzyl halide group), and the coupling agent  147  has a trichlorosilane coupling group. This coupling group is known to couple the silane to a silica surface that has silanol (Si—OH) groups on it, forming Si—O—Si bonds. The resulting surface with linking group  115  includes the chromophore component  145  (i.e., the reactive carbocyclic aromatic linking group) which is attached to the silica surface  100  by a covalent linkage  112 . 
     For other types of surfaces  100 , linking groups  105  having other appropriate coupling agents  147  can be used. For example, polymers have carboxylic acid groups (—COOH) that can react with an alcohol (C—OH) coupling group to form an ester (—COOC) linkage. Surfaces with carboxylic acid groups can also couple with an amino (—NH2) coupling group to form an amide (—CONH—) linkage. Alternatively, carboxylic acid groups on surfaces can be coupled to amine coupling groups to form an amino acid linkage. In other examples, isocyanate coupling groups (—NCO) can be used to form a linkage to surfaces with carboxylic acids, alcohols, and amines. Other reactive functional groups include aromatic chloromethyl, amide, hydrazide, aldehyde, hydroxyl, thiol and epoxy. Clearly, similar reactions can be used for many types of surfaces  100 , as long as the surfaces  100  have functional groups, or can be modified with functional groups, that will react with the coupling agent  147  that is included in the linking group  105 . 
     Returning to a discussion of  FIG. 1 , in a second reaction  125 , a voltage sensitive chromophore precursor  120  is reacted with the surface with linking group  115  to form a monolayer of the voltage sensitive chromophore that is covalently bound to the surface (i.e., voltage sensitive chromophore surface  130 ). The voltage sensitive chromophore surface  130  can also be referred to as the “sensor surface” because the monolayer of the voltage sensitive chromophore provides the function of a sensor for sensing properties of the surface, such as the interfacial electric field, or properties of a liquid that is brought into contact with the surface. 
     In an exemplary embodiment, the voltage sensitive chromophore precursor  120  includes a p-substituted dialkylamino aryl group  180  that is linked to a terminal N-containing heterocyclic aromatic group  185  with a conjugate linkage  182  as illustrated in  FIG. 2B . In this example, the N-containing heterocyclic aromatic group  185  is a pyridinyl group (a pyridinyl group is a radical derived from pyridine). In the second reaction  125 , the chromophore component  145  (i.e., the reactive carbocyclic aromatic linking group) reacts with the terminal N-containing heterocyclic aromatic group  185  of the voltage sensitive chromophore precursor  120  to form the voltage sensitive chromophore  152 . The voltage sensitive chromophore  152  can also be referred to as a voltage sensitive dye (VSD). The voltage sensitive chromophore  152  has an associated dipole moment, and is “voltage sensitive” in that its fluorescence emission spectrum (or its absorbance spectrum) changes when it is placed in an electric field, that is, in a region where the voltage (i.e., potential) is changing. 
     The parts of the voltage sensitive chromophore precursor  120  (i.e., the p-substituted dialkylamino aryl group  180  and the N-containing heterocyclic aromatic group  185 ) are conjugatively linked and are designed to be reacted with the chromophore component  145  to yield the voltage sensitive chromophore  152 . One skilled in the art will recognize that a conjugatively-linked system (also known as a “conjugated system”) is a system of bonded atoms including pi orbitals that have been delocalized through bonding. The pi electrons in these orbitals are delocalized over the atoms that participate in the pi orbital bonding. This normally lowers the total energy of the system, and is favored in such bonding as alternating single and double bonds, certain functional groups attached to aromatic rings, alternating single and double bonds attached to aromatic rings, etc. 
     In alternate embodiments, other types of molecules can be chosen for the two chromophore precursors (i.e., chromophore component  145  and the voltage sensitive chromophore precursor  120 ), depending on the particular voltage sensitive chromophore that is used (e.g., any of the voltage sensitive chromophores in the aforementioned article by P. Fromherz, entitled “Monopole-dipole model for symmetrical solvatochromism of hemicyanine Dyes”). Typically, the resulting voltage sensitive chromophore is a conjugated system, and is formed from any number of alternating single and double bonds and aromatic rings, including functional groups on the aromatic rings. The requirement is that the precursors are hindered from rotation around internal linkages, so as to maintain the largest dipole moment that reverses upon excitation with actinic light, and that the precursors can be conjugatively linked to form the final chromophore. 
     The result of the first and second reactions  110 ,  125  is to provide a monolayer of the voltage sensitive chromophore that is covalently bound to the surface  100  (i.e., the voltage sensitive chromophore surface  130 ). In accordance with the method of  FIG. 1 , a third reaction  150  is also performed to form a voltage sensitive chromophore  152  ( FIG. 2C ), which is dissolved in a liquid  132  to provide a voltage sensitive chromophore solution  155 . 
     As illustrated in  FIG. 2C , in an exemplary embodiment the third reaction  150  is performed by reacting the voltage sensitive chromophore precursor  120  with chromophore component  145  (which is the same chromophore component  145  that was included in the linking group  105  (see  FIG. 2A )), thereby forming the voltage sensitive chromophore  152 . The voltage sensitive chromophore  152  is dissolved in a sample of the liquid  132 , to provide the voltage sensitive chromophore solution  155 . In some cases, the third reaction  150  is carried out in the liquid  132  to directly provide the voltage sensitive chromophore solution  155 . In other cases, the third reaction  150  can be carried out in another solvent, and the voltage sensitive chromophore  152  can then be dried and re-dissolved in the liquid  132  to provide the voltage sensitive chromophore solution  155 . Examples of appropriate liquids  132  that can be used would include water, methanol, ethanol, chloroform, acetone, toluene and methyl chloride. In general, any liquid that does not react chemically with the voltage sensitive chromophore surface  130  to break the covalent bond between the voltage sensitive chromophore  152  and the surface  100  and does not change the conjugated pi system of the voltage sensitive chromophore  152  can be used in accordance with the present invention. For the method of  FIG. 1 , another requirement is that the voltage sensitive chromophore  152  be soluble in the liquid  132 . 
     After, the voltage sensitive chromophore surface  130  and the voltage sensitive chromophore solution  155  have been prepared, fluorescence emission spectra are measured and compared. A measure surface fluorescence emission spectrum step  135  is used to measure a surface fluorescence emission spectrum  140  of the voltage sensitive chromophore surface  130  while it is in contact with a sample of the liquid  132 . This involves irradiating the voltage sensitive chromophore surface  130  with actinic radiation provided by an irradiation source while it is in contact with the liquid and using a fluorescence sensing system to measure the emission spectrum of the resulting fluorescent radiation. In this context, “actinic radiation” is radiation that is adapted to stimulate fluorescence of the voltage sensitive chromophore. Typically, the actinic radiation for many voltage sensitive chromophores will be ultraviolet (UV) radiation or short wavelength visible radiation. For example, in the case of the exemplary voltage sensitive chromophore  152  of  FIGS. 2B-2C , the actinic radiation can be radiation having a wavelength of 480 nm. In some configurations, the liquid  132  can be brought into contact with the voltage sensitive chromophore surface  130  by using a fluid cell (e.g., a cuvette), where the voltage sensitive chromophore surface  130  is used for one or more surfaces of the fluid cell. Similarly, a measure solution fluorescence emission spectrum step  160  is used to measure a solution fluorescence emission spectrum  165  of the voltage sensitive chromophore solution  155 . This can be accomplished by using a fluid cell having transparent windows filled with the voltage sensitive chromophore solution  155 . 
     In some configurations, the solution fluorescence emission spectrum  165  can be predetermined and stored for later comparison with the surface fluorescence emission spectrum  140 . For example, a number of different surfaces  100  can be evaluated using the method of  FIG. 1 . Voltage sensitive chromophore surfaces  130  can be prepared, each having a monolayer of the voltage sensitive chromophore. Surface fluorescence emission spectrum  140  can then be measured for each of the surfaces  100 , and can be compared to the stored solution fluorescence emission spectrum  165  for the voltage sensitive chromophore solution  155  rather than repeating the measurement of the solution fluorescence emission spectrum  165 . 
     Once the surface fluorescence emission spectrum  140  and the solution fluorescence emission spectrum  165  are determined, they are compared using a determine interfacial electric field intensity step  170  to determine a measured interfacial electric field intensity  175 . As will be discussed below, in an exemplary arrangement, the interfacial electric field intensity  175  is determined responsive to a Stark shift in the peaks of the fluorescence emission spectra. 
       FIG. 3A  shows a graph  300  comparing measured fluorescence emission spectra for the case where the exemplary voltage sensitive chromophore  152  of  FIGS. 2B-2C  is used with a quartz surface  100 , and the liquid  132  ( FIG. 1 ) is water. The fluorescence emission spectra were measured using a Spex-JY-Horiba Tau 3 spectrometer with a double monochromator on both the excitation and emission side, and a Peltier-cooled photomultiplier tube detector. To measure the surface fluorescence emission spectrum  140 , a quartz cell (i.e., a cuvette) having a monolayer of the voltage sensitive chromophore  152  on the inside surface was prepared by performing the first and second reactions  110 ,  125  in the cuvette. The cuvette was dried, and then filled with the liquid  132  and irradiated normal to the surface  100  with actinic radiation having an excitation wavelength of 480 nm, and the resulting emission spectrum was measured at an angle of 22°. To measure the solution fluorescence emission spectrum  165 , the voltage sensitive chromophore solution  155  was prepared by dissolving 1 mg of the voltage sensitive chromophore  152  in 15 g of water. This solution was diluted by adding 1 ml of the solution to 19 ml of water (a 1/20 dilution). A cuvette was then filled with the voltage sensitive chromophore solution  155 , and the solution fluorescence emission spectrum  165  was measured as before using the same excitation wavelength (480 nm). 
     It can be seen that the surface fluorescence emission spectrum  140  measured for the voltage sensitive chromophore surface  130  ( FIG. 1 ) having a monolayer of the voltage sensitive chromophore  152  has been shifted toward a higher wavenumber (i.e., to a higher energy level) relative to the solution fluorescence emission spectrum  165  measured for the voltage sensitive chromophore solution  155  ( FIG. 1 ). In this case, the Stark shift between the fluorescence emission spectra (i.e., the change in the wave number between the spectral peeks) was determined to be Δ{tilde over (ν)}=260 cm −1 . 
       FIG. 3B  shows an analogous graph  310  comparing measured fluorescence emission spectra for the case where the liquid is methanol. In this case, the shift between the fluorescence emission spectra was determined to be Δ{tilde over (ν)}=482 cm −1 . Similarly,  FIG. 3C  shows a graph  320  comparing measured fluorescence emission spectra for the case where the liquid is acetone. In this case, the shift between the fluorescence emission spectra was determined to be Δ{tilde over (ν)}=687 cm −1 . 
     Those skilled in the art will recognize that it will typically be desirable to smooth the measured fluorescence emission spectra using an appropriate smoothing algorithm to smooth out any measurement noise. Any appropriate curve smoothing algorithm known in the art can be used in accordance with the present invention. Example curve smoothing algorithms would include low-pass filter algorithms, smoothing spline algorithms, and curve-fitting algorithms that fit a standard functional form (e.g., a Gaussian function) to the measured data. A peak-finding algorithm can then be used to identify the peaks of the fluorescence emission spectra in order to determine the Stark shift. Peak-finding algorithms are well-known in the data processing art, and any appropriate algorithm can be used in accordance with the present invention. 
       FIG. 4  is a schematic showing a molecule of the voltage sensitive dye  152  bonded to a silica surface  100 . The voltage sensitive chromophore  152  has an associated dipole moment {right arrow over (P)} and the surface  100  has an associated interfacial electric field {right arrow over (E)}, with θ being the angle between the {right arrow over (P)} and {right arrow over (E)} vectors. The voltage sensitive dye  152  can rotate around transperiplanar rotomer  340  to align with the {right arrow over (E)} field. 
       FIG. 5A  is a schematic showing a monolayer of the voltage sensitive chromophore  152  having an associated dipole moment {right arrow over (P)} bound to surface  100  in the presence of an interfacial electric field {right arrow over (E)}. Upon illumination by actinic radiation (hν a ), there is a displacement of charge within the voltage sensitive chromophore  152 , causing the dipole moment {right arrow over (P)} to be flipped from ground state  350  to exited state  360 . 
       FIG. 5B  is an energy diagram showing the ground state energy level  352  associated with the ground state  350  ( FIG. 5A ) and the excited state energy level  362  associated with the excited state  360  ( FIG. 5A ). As the voltage sensitive chromophore  152  falls from the exited state  360  back to the ground state  350 , a photon is emitted having an energy ΔE sensor  given by: 
       Δ E   sensor   =hc{tilde over (ν)}   sensor   (1)
 
     where h=6.626×10 −34  J·s is Planck&#39;s constant, c=3.00×10 8  m/s is the speed of light, and {tilde over (ν)} sensor  is the wave number (1/wavelength) of the emitted photon. 
     The excited state energy level  364  associated with the voltage sensitive chromophore  152  when it is in solution is lower than the excited state energy level  362  for the sensor where the voltage sensitive chromophore  152  is bound to the surface  100 , such that the emitted photons have a lower energy ΔE solution  given by: 
       Δ E   solution   =hc{tilde over (ν)}   solution   (2)
 
     where {tilde over (ν)} solution  is the wave number of the emitted photon. 
     The difference between the energies of the excited state energy levels  362 ,  364  (ΔE interface ) corresponds to the Stark shift: 
     
       
         
           
             
               
                 
                   
                     
                       
                         
                           Δ 
                            
                           
                               
                           
                            
                           
                             E 
                             interface 
                           
                         
                         = 
                           
                          
                         
                           
                             Δ 
                              
                             
                                 
                             
                              
                             
                               E 
                               sensor 
                             
                           
                           - 
                           
                             Δ 
                              
                             
                                 
                             
                              
                             
                               E 
                               solution 
                             
                           
                         
                       
                     
                   
                   
                     
                       
                         = 
                           
                          
                         
                           
                             hc 
                              
                             
                               
                                 v 
                                 ~ 
                               
                               sensor 
                             
                           
                           - 
                           
                             hc 
                              
                             
                               
                                 v 
                                 ~ 
                               
                               solution 
                             
                           
                         
                       
                     
                   
                   
                     
                       
                         = 
                           
                          
                         
                           hc 
                            
                           
                               
                           
                            
                           Δ 
                            
                           
                               
                           
                            
                           
                             
                               v 
                               ~ 
                             
                             Stark 
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
     where the Stark shift Δ{tilde over (ν)} Stark ={acute over (ν)} sensor −{tilde over (ν)} solution  is the difference between the wave numbers of the emitted photons in the sensor and solution configurations. The Stark shift can also be related to a corresponding difference in the wavelengths: 
     
       
         
           
             
               
                 
                   
                     
                       
                         
                           Δ 
                            
                           
                               
                           
                            
                           
                             
                               v 
                               ~ 
                             
                             Stark 
                           
                         
                         = 
                           
                          
                         
                           
                             
                               v 
                               ~ 
                             
                             sensor 
                           
                           - 
                           
                             
                               v 
                               ~ 
                             
                             solution 
                           
                         
                       
                     
                   
                   
                     
                       
                         = 
                           
                          
                         
                           
                             1 
                             / 
                             
                               λ 
                               sensor 
                             
                           
                           - 
                           
                             1 
                             / 
                             
                               λ 
                               solution 
                             
                           
                         
                       
                     
                   
                   
                     
                       
                         = 
                           
                          
                         
                           
                             ( 
                             
                               
                                 λ 
                                 solution 
                               
                               - 
                               
                                 λ 
                                 sensor 
                               
                             
                             ) 
                           
                           / 
                           
                             ( 
                             
                               
                                 λ 
                                 sensor 
                               
                               · 
                               
                                 λ 
                                 solution 
                               
                             
                             ) 
                           
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   4 
                   ) 
                 
               
             
           
         
       
     
     where λ sensor  and λ solution  are the wavelengths of the emitted photons in the sensor and solution configurations, respectively. 
     The energy difference ΔE interface  corresponds to the energy difference between the two orientations of the chromophore dipole in the presence of the interfacial electric field, which is directly related to the magnitude of interfacial electric field {right arrow over (E)} and the chromophore dipole moment {right arrow over (P)}, and the angle θ between them: 
       Δ E   interface =2| {right arrow over (E)}∥{right arrow over (P)} |cos θ  (5)
 
     Combining Eqs. (3) and (5) and solving for the magnitude of the interfacial electric field {right arrow over (E)} gives: 
     
       
         
           
             
               
                 
                   
                      
                     
                       E 
                       -&gt; 
                     
                      
                   
                   = 
                   
                     
                       hc 
                        
                       
                           
                       
                        
                       Δ 
                        
                       
                           
                       
                        
                       
                         
                           v 
                           ~ 
                         
                         Stark 
                       
                     
                     
                       2 
                        
                       
                          
                         
                           P 
                           -&gt; 
                         
                          
                       
                        
                       cos 
                        
                       
                           
                       
                        
                       θ 
                     
                   
                 
               
               
                 
                   ( 
                   6 
                   ) 
                 
               
             
           
         
       
     
     Therefore, experimentally measuring the Stark shift Δ{tilde over (ν)} Stark  enables the magnitude of the interfacial electric field |{right arrow over (E)}| to be determined given estimates of the dipole moment and the angle that the chromophore dipole makes with the interfacial electric field at the interface. The cosine function is relatively close to 1.0 for a fairly large range of angles around θ=0°, so that it can be neglected in many cases without significantly impacting the calculated result. Even if θ is as large as 60°, the calculation of the electric field would only be in error by a factor of about 2×. Note that any solvatochromic shift associated with the particular liquid is nulled out given that both spectra are taken in the same liquid. 
     The dipole moment of the voltage sensitive chromophore  152  of  FIGS. 2B-2C , which was used to produce the experimental results of  FIGS. 3A-3C  was estimated to be: 
         {right arrow over (P)}=e·δ/ 2=(1.60×10 −19  C)(0.217 nm)/2=1.74×10 −29  C·m  (7)
 
     where e=1.60×10 −19  C is the elementary charge, and δ=0.217 nm is the estimated distance that the charge is displaced when the polarity of the dipole is flipped. Using this value, the magnitudes of the interfacial electric fields for each case can be calculated using Eq. (6). The results are summarized in Table 1, where the dipole angle was estimated to be θ=45°. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Measured interfacial electric fields 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Surface 
                 Liquid 
                 Δ{tilde over (v)} (cm −1 ) 
                 |{right arrow over (E)}| (V/cm) 
               
               
                   
                   
               
               
                   
                 quartz 
                 water 
                 260 
                 2.11 × 10 6   
               
               
                   
                 quartz 
                 methanol 
                 482 
                 3.90 × 10 6   
               
               
                   
                 quartz 
                 acetone 
                 687 
                 5.56 × 10 6   
               
               
                   
                   
               
            
           
         
       
     
     From Table 1, it can be seen that for a given surface (e.g., quartz with a monolayer of a particular voltage sensitive chromophore  152 ), the magnitude of the interfacial electric field, and therefore the Stark shift, vary according to the characteristics of the liquid. In some embodiments this fact can be used to provide a sensor for determining characteristics of an unknown liquid. One of the primary liquid characteristics that will influence the interfacial electric field is the dielectric constant of the liquid.  FIG. 6  shows a graph  400  of the Stark shift Δ{tilde over (ν)} Stark  as a function of the dielectric constant, ∈ r , using the data from Table 1. A curve  410  has been fit to the measured data, giving a relationship between the dielectric constant and the resulting Stark shift. If the Stark shift is measured for an unknown liquid, the curve  410  can be used to determine an estimate of the dielectric constant of the liquid. 
       FIG. 7  shows a flow chart of a method for determining a characteristic of a liquid in accordance with an embodiment of the invention. The method of  FIG. 7  is identical to that of  FIG. 1 , except that rather than analyzing the surface and solution fluorescence emission spectra  140 ,  165  to determine the interfacial electric field intensity  175 , a determine characteristic of liquid step  200  is used to determine a liquid characteristic  205 . 
     In an exemplary arrangement, the determine characteristic of liquid step  200  computes the Stark shift, and then uses the curve of  FIG. 6  to determine a dielectric constant of the liquid  132 . For the case where the liquid  132  is an unknown substance, the determine characteristic of liquid step  200  can be used to identify the liquid, or to determine one or more candidate identities for the liquid. For example, a table of dielectric constants for known liquids can be prepared, and the measured dielectric constant for the unknown liquid can be compared to the table to identify candidate liquids having dielectric constants that match the measured dielectric constant to within the associated measurement error. This approach can be used to construct sensors capable of identifying or characterizing liquids without the use of complexing agents or selective membranes as is generally required in the prior art. Other examples of liquid characteristics  205  that could be determined in a similar fashion in accordance with the invention would include the polarizability and electric susceptibility. 
     In some embodiments, the determine characteristic of liquid step  200  determines the liquid characteristic  205  based on only the surface fluorescence emission spectrum  140 , without measuring the solution fluorescence emission spectrum  165 . For example, surface fluorescence emission spectra  140  can be measured for a library of common liquids using the voltage sensitive chromophore surface  130 . A surface fluorescence emission spectrum  140  can then be measured for an unknown liquid  132 . The resulting surface fluorescence emission spectrum  140  for the unknown liquid can then be compared to the library of surface fluorescence emission spectra  140  for the common liquids to determine whether there is a match. If so, it can be determined that the unknown liquid is likely to be the matching liquid from the library. In this example, the determined characteristic  205  is the identity of the liquid  132 . 
       FIG. 8  shows a flow chart of a method for determining a characteristic difference between different liquids in accordance with an embodiment of the invention. The method of  FIG. 8  is similar to that of  FIG. 1 , except that rather than comparing the fluorescence emission spectrum determined from the voltage sensitive chromophore surface  130  with that measured for the voltage sensitive chromophore solution  155 , the voltage sensitive chromophore surface  130  is used to compare a first liquid  210  and a second liquid  215 . In this case, a measure first fluorescence emission spectrum step  220  provides a first fluorescence emission spectrum  225  for the first liquid  210  in contact with voltage sensitive chromophore surface  130 , and a measure second fluorescence emission spectrum step  230  provides a second fluorescence emission spectrum  235  for the second liquid  215  in contact with the voltage sensitive chromophore surface  130 . A characterize difference between liquids step  240  is then used to compare the first and second fluorescence emission spectra  225 ,  235  to determine a difference between the liquids  245 . For example, the difference between the liquids  245  can be a parameter representing the degree of similarity (or difference) between the first and second fluorescence emission spectra  225 ,  235  (e.g., a correlation coefficient, or an RMS difference between the spectra). Alternatively, the difference between the liquids  245  can be a parameter indicating the likelihood that the first liquid  210  and the second liquid  215  are the same (e.g., a statistical confidence level). In other arrangements, the difference between the liquids  245  can be one or more parameters characterizing the difference between the first and second fluorescence emission spectra  225 ,  235  (e.g., a difference between the peak wavelengths, or a difference function representing the difference as a function of wavelength). 
     A variety of applications can be envisioned for the method of  FIG. 8 . For example, a distillation process can be used to extract a particular liquid from a mixture of liquids. During the distillation process, the purity of the liquid will gradually increase. In this case, the first liquid  210  can be a reference sample of liquid having a desired level of purity, and the second liquid  215  can be a sample of the distillation product at a particular point in time. The difference between the liquids  245  can be determined at a series of times, and the distillation process can be terminated when it is determined that the difference between the liquids  245  falls below a predefined threshold indicating that the distillation product matches the reference sample. 
     While the method of  FIG. 8  was described with respect to comparing first and second liquids  210 ,  215 , one skilled in the art will recognize that the method can be generalized to compare two fluids, where one or both of the fluids can be gases. 
     The determine interfacial electric field intensity step  170  of  FIG. 1 , the determine characteristic of liquid step  200  of  FIG. 7  and the characterize difference between liquids step  240  of  FIG. 8  generally involve making a comparison between a pair of fluorescence emission spectra. The comparison methods described above have involved determining a Stark shift by comparing the positions of the peaks of the fluorescence emission spectra (either in terms of wave number differences or wavelength differences). In other arrangements, different methods can be used to compare the two fluorescence emission spectra. For example, in one variation, rather than comparing the peaks of the fluorescence emission spectra, centroids (or other central tendency metrics) can be determined for each fluorescence emission spectra and compared. Other statistical metrics can also be used to characterize the fluorescence emission spectra, including standard deviation, skew and kurtosis. 
       FIG. 9  is a graph illustrating exemplary first and second fluorescence emission spectra  225 ,  235 . In this case, the first surface fluorescence emission spectrum  225  was determined for a first liquid  210  (water), and the second fluorescence emission spectrum  225  was determined for a second liquid  215  (methanol), in accordance with the method of  FIG. 8 . 
     In an exemplary class of spectrum comparison methods, a first wavelength  430  and a second wavelength  435  are defined. In the illustrated example, the first wavelength  430  is 650 nm and the second wavelength  435  is 560 nm. Preferably, the first and second fluorescence emission spectra  225 ,  235  overlap with both the first and second wavelengths  430 ,  435 . A first intensity ratio R 1 =A 1 /B 1  is determined for the first fluorescence emission spectrum  225 , where A 1  is the value of the first fluorescence emission spectrum  225  at the first wavelength  430 , and B 1  is the value of the first fluorescence emission spectrum  225  at the second wavelength  435 . Likewise, a second intensity ratio R 2 =A 2 /B 2  is determined for the second fluorescence emission spectrum  235 , where A 2  is the value of the second fluorescence emission spectrum  235  at the first wavelength  430 , and B 2  is the value of the second fluorescence emission spectrum  235  at the second wavelength  435 . 
     If a fluorescence emission spectrum is centered between the first and second wavelengths  430 ,  435 , then the intensity ratio will be approximately equal to 1.0. If the spectrum is to the left of center, then the intensity ratio will be smaller than 1.0, and if the spectrum is to the right of center, then the intensity ratio will be greater than 1.0. Therefore, the intensity ratio will be a measure of the location of the spectral peak for the spectrum, and the difference in the intensity ratios will be a measure of the shift in the spectral peaks. 
     Table 2 shows exemplary ratios determined for the fluorescence emission spectra of  FIG. 9 . It can be seen that the difference between spectral peaks is clearly reflected by differences in the intensity ratios. The larger intensity ratio for water reflects the fact that its emission spectrum (first fluorescence emission spectrum  225 ) is shifted to the right relative to that for methanol (second fluorescence emission spectrum  235 ). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Intensity ratio comparison 
               
            
           
           
               
               
               
               
            
               
                   
                 A (λ = 650 nm) 
                 B (λ = 560 nm) 
                 R = A/B 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 Water 
                 0.680 
                 0.349 
                 1.95 
               
               
                 Methanol 
                 0.510 
                 0.690 
                 0.74 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the intensity ratios can be related to the quantity being measured (e.g., the interfacial electric field intensity  175  ( FIG. 1 ) or the liquid characteristic  205  ( FIG. 7 )) by evaluating the intensity ratios for a set of reference configurations and fitting a function to the results. 
     In other types of spectrum comparison methods more than two wavelengths can be compared. For example, additional spectra ratios can be computed between additional wavelengths pairs to provide additional information about the shape and position of the spectrum. In some cases, a spectral difference can be determined by subtracting the two spectra on a wavelength-by-wavelength basis. The spectral difference can then be analyzed to characterize the difference between the spectra. For example, an RMS spectral difference can be computed between the spectra measured for two liquids to determine if the spectra are statistically indistinguishable, indicating that the two liquids may be the same. In some configurations, the spectra can be analyzed to determine corresponding color values (e.g., in the well-known L*a*b* color space), and color differences can be computed (e.g., ΔE* values) to characterize the difference between two spectra. 
     In some embodiments, many spectra are compared to determine which liquid is present at the surface. In such cases, any classification and discrimination analysis methods known in the statistics and chemometrics fields can be used. These methods include the use of the Mahalanobis distance, K nearest neighbor, discriminant analysis, cluster analysis, factor analysis, and principal component analysis (PCA) and obtaining spectral contrast angles between pairs of spectra. As the number of compared spectra increases, the more useful these methods become, especially PCA and cluster analysis, leading to full pattern recognition of the spectral shape of a given liquid/chromophore spectral response. Many of these techniques can be extended to spectral modeling or deconvolution, allowing a liquid to be identified by all of its wavelength responses. 
       FIG. 10  shows an exemplary surface evaluation system  500  for evaluating a surface. A chromophore application system (not shown) is used to apply a monolayer of a voltage sensitive chromophore  152  ( FIG. 2B ) to a surface  100  ( FIG. 2B ) to provide voltage sensitive chromophore surface  130 . The applied voltage sensitive chromophore  152  is covalently bound to the surface  100  and has a fluorescence emission spectrum which varies in accordance with a characteristic of the surface  100  (e.g., the interfacial electric field). The voltage sensitive chromophore surface  130  is mounted in a surface holder  505  for measurement. One skilled in the art will recognize that a wide variety of arrangements can be used for the surface holder  505  in accordance with the present invention. In the illustrated configuration, the surface holder  505  includes a recessed surface that is adapted to receive a liquid  132  in some embodiments. In other configurations, the voltage sensitive chromophore surface  130  can be a surface of a liquid cell (e.g., a cuvette), and the surface holder  505  can be a mechanism for holding the liquid cell. 
     An irradiation source  510  is positioned to irradiate the voltage sensitive chromophore surface  130  with actinic radiation that stimulates fluorescence of the voltage sensitive chromophore  152 . A fluorescence sensing system  515  (e.g., a spectrophotometer) measures a surface fluorescence emission spectrum  140  which is emitted by the irradiated voltage sensitive chromophore surface  130 . In some embodiments, the fluorescence sensing system  515  is a micro-spectrophotometer which is adapted to sense the surface fluorescence emission spectrum  140  emitted from a localized area voltage sensitive chromophore surface  130 . In this case, the irradiation source  510  can illuminate the same localized area (as shown in  FIG. 10 ), or alternatively can illuminate a broad area of the voltage sensitive chromophore surface  130 . 
     Generally, the surface fluorescence emission spectrum  140  will be measured while the voltage sensitive chromophore surface  130  is in contact with some fluid (e.g., a liquid or a gas). In some embodiments, the surface fluorescence emission spectrum  140  can be measured while the voltage sensitive chromophore surface  130  is exposed to air (or some other gas). In other embodiments, the surface fluorescence emission spectrum  140  can be measured while the voltage sensitive chromophore surface  130  is in contact with a liquid  132 . In this case, a liquid applicator (not shown) can be used to bring the liquid  132  into contact with the voltage sensitive chromophore surface  130 . For example, the liquid  130  can be added into the recessed region in the surface holder  505  of  FIG. 10 . For the case where the voltage sensitive chromophore surface  130  is the wall of a liquid cell, the liquid applicator can add the liquid  132  to the liquid cell. Depending on the geometry of the voltage sensitive chromophore surface  130  and the other components of the surface evaluation system  500 , one skilled in the art will recognize that a wide variety of liquid applicators can be used to bring the liquid  132  into contact with the voltage sensitive chromophore surface  130 . 
     An analysis system  520  is used to analyze the surface fluorescence emission spectrum  140  to evaluate a characteristic of the voltage sensitive chromophore surface  130 . In an exemplary embodiment, the characteristic is the interfacial electric field intensity  175  ( FIG. 1 ) of the voltage sensitive chromophore surface  130 . The analysis steps involved with the determination of the interfacial electric field intensity  175  have been discussed above. In some embodiments, the solution fluorescence emission spectrum  165  of the voltage sensitive chromophore solution  155  can be predetermined and stored as a reference fluorescence emission spectrum for comparison with the measured surface fluorescence emission spectrum  140  at a later time. In other embodiments, the analysis system  520  can be used to determine a liquid characteristic  205  as discussed with respect to  FIG. 7 , or to determine differences between a plurality of liquids, as was discussed with respect to  FIG. 8 . 
     In some embodiments, the fluorescence sensing system  515  measures a plurality of surface fluorescence emission spectra  140  at a lattice of spatial positions  525  on the voltage sensitive chromophore surface  130 . In this case, either the surface holder  505  or the fluorescence sensing system  515  can be moved in a scanning path in order to sense the surface fluorescence emission spectra  140  at the different positions. In such configurations, the analysis system  520  can be used to analyze the plurality of surface fluorescence emission spectra  140  to evaluate a uniformity of a characteristic of the voltage sensitive chromophore surface  130 . This can be useful for a number of applications, including detecting surface defects or inspecting features on the surface. 
     There are a wide variety of applications that involve depositing a pattern of features onto a surface, including the fabrication of electrical components or devices (e.g., on a silicon wafer or on a glass or polymer substrate). In some cases, it can be difficult to inspect such components, particularly when they may include features that are fabricated using transparent, or partially transparent, materials. Such features may not be easily detected by conventional machine vision inspection techniques that involve capturing an image of the surface using visible light. However, the presence of the features will produce local perturbations in the interfacial electric field of the surface. These perturbations can be detected using methods in accordance with embodiments of the present invention by applying a monolayer of voltage sensitive chromophore to the surface and measuring the fluorescence emission spectrum. The presence of edges and surface textures on the surface can also affect the amplitude and direction of the emitted fluorescent light. Such perturbations can be detectable with appropriate types of sensors. 
     In one such embodiment, the surface  100  having the features thereon is treated to attach a monolayer of the voltage sensitive chromophore  152  ( FIG. 2B ) providing the voltage sensitive chromophore surface  130  as described above relative to  FIG. 1 . The surface evaluation system  500  can then be used to measure a pattern of fluorescent light as a function of spatial position on the voltage sensitive chromophore surface  130 . In an exemplary configuration, the pattern of fluorescent light can be characterized by measuring the surface fluorescence emission spectra  140  at a lattice of spatial positions  525  as described above. In other configurations, the pattern of fluorescent light can be characterized by measuring an intensity or a color (or some other attribute) of the fluorescent light. In such cases, the fluorescence sensing system  515  does not necessarily need to determine the surface fluorescence emission spectra  140 , but rather can directly measure the fluorescent light characteristic using other types of measurements (e.g., intensity measurements or color measurements). 
     The analysis system  520  can analyze the measured pattern of fluorescent light in order to inspect the surface. In some embodiments, features in the measured pattern of fluorescent light can be compared to a reference pattern associated with the device being fabricated, for example to verify that there are no shorts or voids in a pattern of micro-wires. In some embodiments, the measured pattern of fluorescent light can be analyzed to detect surface defects (e.g., non-uniformities in a surface region that was supposed to be uniform). 
     When the surface evaluation system  500  is being used to measure a pattern of fluorescent light, it may not be necessary to measure the fluorescent light while the voltage sensitive chromophore surface  130  is in contact with a liquid  132 , particularly if the goal is to detect non-uniformities or other defects rather than determining quantitative interfacial electric field intensities. In such cases, the pattern of fluorescent light can be measured when the voltage sensitive chromophore surface  130  is exposed to air (or some other gas). 
     The methods described herein have many advantages compared to other methods for using voltage sensitive chromophores currently used in the art. The describes methods provide a chromophore that is covalently attached to the surface. In contrast, other methods, such as those described in the aforementioned articles by Loew and Pope et al., use chromophores embedded in membranes or self-assembled monolayers. In such arrangements, the location of the chromophore is not known precisely, nor is the measured fluorescence emission spectra guaranteed to be from a chromophore in a known position with respect to the surface or membrane, but can be mixed with the spectra of unembedded chromophore molecules and chromophore molecules at various distances from the surface. In the methods of the present invention, the voltage sensitive chromophore is attached to the surface in a known way, and is created only when it is attached to the surface, so the position of the chromophore is known precisely. 
     Another advantage of the present invention is that it can be used with a wide variety of different surfaces including silica, polymer, metal, inorganic, and other surfaces. Other methods have restrictions on the sample surface, such as requiring a biological membrane a metal surface or a metal electrode. For example, the method described in the aforementioned articles by Pope et al. require that the surface be a metal electrode, and specifically a silver electrode, in order to enhance the fluorescence of the chromophore. Our method has been shown to work without metal-enhanced fluorescence. 
     In other methods, such as those described in the aforementioned articles by Pope et al., external fields need to be applied (for example, to an electrode surface or a membrane surface) to provide a change in the electric field to induce a Stark shift, which is measured by a shift in the fluorescence emission spectrum. In methods of the present invention, the emission of the chromophore on the surface is compared to the emission of the chromophore in solution, which enables the Stark shift to be determined and the interfacial electric field to be calculated without the use of any applied field. This makes the measurement dependent only on the voltage sensitive chromophore, the surface, and the liquid, and is not dependent on any applied fields. 
     Because the methods in this application do not require applied fields, the methods are simpler and do not require the use of more complicated instrumentation. Additionally, the methods can be applied over a large area, and need not be confined to smaller areas, such as in atomic force microscopy. 
     Furthermore, the methods are non-destructive for the sample, and only require that the surface be modified to include the voltage sensitive chromophore. In other methods for determining an interfacial electric field, such as the streaming potential method described in the aforementioned article by Xie et al. and the atomic force microscopy method described in the aforementioned article by Li et al., the sample is significantly changed, and may not be used for any other testing. 
     Generally, the methods of the present invention are simpler, more precise, more accurate, and more versatile than prior art methods for measuring interfacial electric fields. Furthermore, the present methods can be used to characterize large areas of a surface, and to provide a sensor for characterizing a liquid. 
     The invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention. 
     PARTS LIST 
     
         
           100  surface 
           105  linking group 
           110  first reaction 
           112  covalent linkage 
           115  surface with linking group 
           120  voltage sensitive chromophore precursor 
           125  second reaction 
           130  voltage sensitive chromophore surface 
           132  liquid 
           135  measure surface fluorescence emission spectrum step 
           140  surface fluorescence emission spectrum 
           145  chromophore component 
           147  coupling agent 
           150  third reaction 
           152  voltage sensitive chromophore 
           155  voltage sensitive chromophore solution 
           160  measure solution fluorescence emission spectrum step 
           165  solution fluorescence emission spectrum 
           170  determine interfacial electric field intensity step 
           175  interfacial electric field intensity 
           180  p-substituted dialkylamino aryl group 
           182  conjugate linkage 
           185  N-containing heterocyclic aromatic group 
           200  determine characteristic of liquid step 
           205  liquid characteristic 
           210  first liquid 
           215  second liquid 
           220  measure first fluorescence emission spectrum step 
           225  first fluorescence emission spectrum 
           230  measure second fluorescence emission spectrum step 
           235  second fluorescence emission spectrum 
           240  characterize difference between liquids step 
           245  difference between the liquids 
           300  graph 
           310  graph 
           320  graph 
           340  transperiplanar rotomer 
           350  ground state 
           352  ground state energy level 
           360  exited state 
           362  exited state energy level 
           364  exited solution state energy level 
           400  graph 
           410  curve 
           420  graph 
           430  first wavelength 
           435  second wavelength 
           500  surface evaluation system 
           505  surface holder 
           510  irradiation source 
           515  fluorescence sensing system 
           520  analysis system 
           525  lattice of spatial positions