Patent Publication Number: US-11035783-B2

Title: Normalizing the response of a fluorescence instrument using spectral response

Description:
CROSS REFERENCE TO RELATED APPLICATION(S) 
     This application is a continuation of U.S. patent application Ser. No. 15/512,343, filed Mar. 17, 2017, which is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/US2015/050576, filed Sep. 17, 2015, which claims priority under applicable portions of 35 U.S.C. § 119 of U.S. Patent Application Ser. No. 62/052,132, filed Sep. 18, 2014, the entire contents of each application being incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention relates generally to a method of calibrating diagnostic analyzers using fluorometry as a measurement mechanism. 
     BACKGROUND OF THE INVENTION 
     The present invention pertains to at least one clinical diagnostic analyzer conducting an immunoassay employing a fluorescence label. Typically, a fluorescence label is bound to antibodies or antigens having an affinity for the analyte of interest. The unknown analyte in the sample then binds with the labeled antibodies or antigens which are usually immobilized to a substrate. The unbound, labeled antibodies or antigens are subsequently washed away, and the concentration of bound, labeled antibodies or antigens is measured using fluorometry. 
     Fluorometry is the measurement of fluorescence. Fluorescence is the molecular adsorption of light energy at one wavelength and its nearly instantaneous re-emission at another, usually longer, wavelength. The instrument used to measure fluorescence is called a fluorometer. A fluorometer generates the wavelength of light required to excite the analyte of interest and then it measures the intensity of the resulting emitted light. The amount or quantity of emitted light is frequently proportional to the concentration of the analyte being measured. When employed in clinical diagnostic analyzers fluorometry provides extraordinary sensitivity, high specificity, simplicity, and low cost as compared to other analytical techniques. 
     To insure the quality control of results from fluorometers, some form of stable reference standard is employed such as National Institute of Standards and Technology (NIST) SRM (Standard Reference Material) 2944 glass. SRM 2944 is a cuvette-shaped, bismuth-ion-doped glass, recommended for use for relative spectral correction of emission and day-to-day performance verification of steady-state fluorescence spectrometers. Further information regarding SRM 2944 is described by Paul C. DeRose; Melody V. Smith; Jeffrey R. Anderson; Gary W. Kramer in the Journal of Luminescence, Volume 141, pp. 9-14, entitled “Characterization of Standard Reference Material 2944, Bi-Ion-Doped Glass, Spectral Correction Standard for Red Fluorescence” which is hereby incorporated by reference in its entirety. 
     One problem presented by fluorometers is that variations in the manufacture of clinical diagnostic analyzers are such that for a given fluorescence label; the population of clinical diagnostic analyzers will not provide the same analytical result for a specific quantity of analyte in a sample. These manufacturing variations result from differences in excitation light spectra from the laser diode, variances in transmission characteristics of optical filters, etc. Hence, to account for these variations and to provide accurate results each individual clinical diagnostic analyzer must be calibrated. 
     Another problem presented by fluorometers is that the introduction of a new fluorescence label having differing absorption and emission spectra will require a total re-calibration of the entire clinical diagnostic analyzer population. 
     SUMMARY OF THE INVENTION 
     One object of the present invention is to enable a population of clinical diagnostic analyzers or instruments to be normalized to a specific master clinical diagnostic analyzer or instrument such that the response of any subordinate clinical diagnostic analyzer or instrument in the population to a sample having a specific amount of analyte is substantially the same as the response of the master clinical diagnostic analyzer or instrument to that sample after an initial factory calibration. 
     Another object of the present invention is allow the introduction of a new fluorescence label having a different adsorption and emission spectrum as compared to a prior fluorescence label such that the re-calibration and re-normalization of the entire population of clinical diagnostic analyzers to the master clinical diagnostic analyzer depends only upon the absorption and emission spectra of the new fluorescence label. Total re-calibration of the population of clinical diagnostic analyzers is not required. 
     The foregoing and further objects of the invention are accomplished according to one aspect of the invention that provides a method of normalizing a first diagnostic result of a subordinate clinical diagnostic analyzer to a second diagnostic result of a master clinical diagnostic analyzer comprising the steps of obtaining a normalized excitation intensity spectrum of the master clinical diagnostic analyzer, obtaining a normalized excitation intensity spectrum of the subordinate clinical diagnostic analyzer, obtaining a normalized responsivity intensity spectrum of the master clinical diagnostic analyzer, obtaining a normalized responsivity intensity spectrum of the subordinate clinical diagnostic analyzer, obtaining a normalized excitation/emission spectrum of a solid inorganic photostable fluorophore calibration target, reading the solid inorganic photostable fluorophore calibration target in the master clinical diagnostic analyzer thereby obtaining a first response value, reading the solid inorganic photostable fluorophore calibration target in the subordinate clinical diagnostic analyzer thereby obtaining a second response value, determining the gain ratio of the master clinical diagnostic analyzer to the subordinate clinical diagnostic analyzer based upon the two above obtained response values, determining a multiplicative normalization factor between a normalized subordinate clinical diagnostic analyzer and the master clinical diagnostic analyzer, determining the relative adsorption/emission spectrum of a first fluorescently labeled dye whereas the first fluorescently labeled dye is a diagnostic assay component, obtaining a first diagnostic result from a specific patient specimen or sample incorporating the first fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer, and multiplying the first diagnostic result by the multiplicative normalization factor to obtain a second diagnostic result whereas the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer. 
     Still another aspect of the invention provides a method to re-normalize a subordinate clinical diagnostic analyzer assay result as compared to a master clinical diagnostic analyzer assay result comprising the steps of normalizing the subordinate clinical diagnostic analyzer as above, obtaining a relative adsorption/intensity spectrum of a second fluorescently labeled dye whereas the second fluorescently labeled dye is a diagnostic assay component, determining a re-normalization multiplicative factor between a subordinate clinical diagnostic analyzer and a master clinical diagnostic analyzer, obtaining a first diagnostic result from a specific patient specimen or sample incorporating the second fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer, and multiplying the first diagnostic result by the re-normalization factor to obtain a second diagnostic result whereas the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer. 
     Further objects, features and advantages of the present invention will be apparent to those skilled in the art from detailed consideration of the preferred embodiments that follow. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a view of the chemistry associated with an immunoassay usually contained in an analytic slide (not shown). 
         FIG. 2  is a schematic diagram of an optical detection system associated with a fluorescence detection analyzer. 
         FIG. 3  is a graph of the relative adsorption and emission spectrum of Alexa Fluor® 635, a dye commonly used a fluorophore label. 
         FIG. 4  is a graph of the relative adsorption and emission spectrum of Alexa Fluor® 647, another dye commonly used a fluorophore label. 
         FIG. 5  is a graph of an example master instrument linear calibration curve. 
         FIG. 6  is a graph of an example master instrument nonlinear calibration curve. 
         FIG. 7  is a histogram of the responses to a set of fixed analyte concentration samples or specimens using Alexa Fluor® 647 when subjected to instrument variations. 
         FIG. 8  is a histogram of the ratio of responses to a set of fixed analyte concentration samples or specimens comparing bismuth-doped phosphor glass and Alexa Fluor® 647 when subjected to instrument variations. 
         FIG. 9  is a histogram of the ratio of responses to a set of fixed analyte concentration samples or specimens comparing bismuth-doped phosphor glass and Alexa Fluor® 647 when subjected to instrument variations when corrected using instrument normalization. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     While the present invention is described with respect to preferred embodiments as detailed below and shown in the figures, the present invention is limited only by the metes and bounds of the claims that follow. 
     Fluorometry is chosen for its extraordinary sensitivity, high specificity, simplicity, and low cost as compared to other analytical techniques. Fluorometry is ordinarily 1000-fold more sensitive than absorbance measurements. It is a widely accepted and powerful technique that is used for a variety of environmental, industrial, and biotechnology applications. It is a valuable analytical tool for both quantitative and qualitative analysis. However, fluorometry requires a stable fluorescence standard to insure that the clinical diagnostic analyzers remain normalized and in calibration. Organic fluorophores, especially those in liquid form, are not well suited for use in normalizing analyzers in a factory setting because they photobleach, have limited shelf life, are prone to carryover problems, and are difficult to dose. A solid inorganic photostable fluorophore would not have the above problems, but there are only a limited number of these materials available. 
     In conjunction with a preferred embodiment of the present inventive method, a device has been designed using a National Institute of Standards and Technology (NIST) developed material consisting of a phosphate matrix glass doped with bismuth ion such that the glass has fluorescent properties. This material is known as NIST Standard Reference Material (SRM) 2944 glass. The composition of such material is shown in Table A. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Composition of NIST SRM 2944 Glass 
               
            
           
           
               
               
               
               
            
               
                   
                 Ingredient 
                 Weight % 
                 Mole % 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 P 2 O 5   
                 70.52 
                 49.84 
               
               
                   
                 CaO 
                 26.20 
                 47.05 
               
               
                   
                 Al 2 O 3   
                 3.04 
                 3.00 
               
               
                   
                 Bi 2 O 3   
                 0.51 
                 0.11 
               
               
                   
                   
               
            
           
         
       
     
     This device has been designed to overcome the limitations listed above and is used in connection with this inventive method; see copending United States patent application by Freeman III, Heavner, and Oenick entitled “Fluorescence Reference Standard Device” (Application No. PCT/US15/50608) which is hereby incorporated by reference in its entirety. For different wavelength fluorometry a different material other than NIST SRM 2944 would be used, such as other phosphate doped glasses also available from NIST, including SRM 2943, copper doped glass, spectral correction standard for blue fluorescence. 
     The above described NIST SRM 2944 glass device is a preferred solid inorganic photostable fluorophore used in the inventive method described herein to normalize a population of subordinate clinical diagnostic analyzers to a master clinical diagnostic analyzer. Using the excitation and emission spectrums of the NIST SRM 2944 glass, the excitation and emission spectrums of the fluorescence label employed in the combination fluorescently labeled label antibody reagent, and the measured excitation and responsivity spectrums inherent in the optical detection systems of the master and subordinate clinical diagnostic analyzers, normalization of subordinate analyzers to the master analyzer is performed at the factory. Using samples or specimens of known analyte concentrations, a standard calibration can likewise be performed at the factory. And furthermore, should it be required or desirable to change the label in the combination fluorescence label antibody reagent, this can be accomplished in the field using only the excitation and emission spectrums of the new fluorescence label. 
     One advantage of the inventive method is that by using solid inorganic photostable fluorophore, such as the preferred NIST SRM 2944 glass, as a reference material, a population of subordinate clinical diagnostic analyzers can be normalized to one master clinical diagnostic analyzer such that after a factory normalization and calibration the subordinate clinical diagnostic analyzers will have substantially the same response to a sample or specimen containing a fixed amount of analyte as would the master clinical diagnostic analyzer. 
     Furthermore, should it become necessary or desirable to change the fluorescence label in the combination fluorescence label antibody reagent, then the population of subordinate clinical diagnostic analyzers can be re-normalized (and retain the original factory calibration) by a simple procedure not requiring a total recalibration in the field. 
     For a general understanding of the disclosed methods, reference is made to the drawings. In the drawings, like reference numerals have been used to designate identical elements. In describing the disclosed methods, the following term(s) have been used in the description. 
     The term “ξ” (the Greek letter xi) or “emission” refers to one or more wavelengths of light generated as a result of fluorescence, specifically when “4” is used in an equation it stands for emission wavelength. 
     The term “responsivity” refers to the normalized output of an optical intensity measuring system as a function of a specific wavelength of light being input to that system. 
     The term “χ” (the Greek letter chi) or “excitation” refers to one or more wavelengths of light generated to be used as a source to radiate a fluorescence complex, specifically when “χ” is used in an equation it stands for excitation wavelength. 
     The term “absorbance” refers to the normalized extinction coefficient of a fluorescent dye. 
     The term “spectral distribution” or “shape function” refers to the relative intensity of an excitation or emission light beam as a function of wavelength. 
     The term “clinical diagnostic analyzer,” “diagnostic analyzer,” and “instrument” are taken to mean devices that accept a patient sample or specimen, analyze the sample or specimen for a specific analyte, and report the result of that analysis. These terms are meant to encompass clinical chemistry analyzers, immunohematology analyzers, lateral flow device readers, and the like. 
     The term “normalize” refers to the inventive method applied to two clinical diagnostic analyzers or instruments, a master instrument “A” and a subordinate instrument “B”, such that the response of “B” to a specific sample or specimen containing a certain concentration of analyte can be converted to the response of “A” to the same sample or specimen by using a multiplicative factor when the assay method employed by the analyzers uses a common fluorescently labeled dyes. 
     The term “re-normalize” refers to the inventive method applied to two clinical diagnostic analyzers or instruments, a master instrument “A” and a subordinate instrument “B”, such that the response of “B” to a specific sample or specimen containing a certain concentration of analyte can be converted to the response of “A” to the same sample or specimen by using a multiplicative factor when the assay method employed by the analyzers uses differing fluorescently labeled dyes. 
     The terms “Alexa Fluor® 635” and “Alexa Fluor® 647” refer to preferred organic fluorophores that can be used as fluorescent tags. These materials are made by INVITRO-GEN™. For example, the adsorption/emission spectrum of “Alexa Fluor® 635” is shown in  FIG. 3  and “Alexa Fluor® 647” has a absorption maximum at 650 nm and an emission maximum at 671 nm as shown in  FIG. 4 . “Alexa Fluor® 635” is sometimes abbreviated as “AF 635” and “Alexa Fluor® 647” is sometimes abbreviated as “AF 647”. 
     In  FIG. 1  a combination fluorescently labeled antibody reagent  101  is added to a target analyte  102  (an antigen in this specific example) in the sample or specimen wherein the combination fluorescence label antibody reagent  101  binds to the analyte forming an antibody-analyte complex  103 . Unbound combination fluorescence label antibody reagent  101  is subsequently removed. The bound antibody-analyte complex  103  is then exposed to an excitation light of specific wavelength causing a fluorescence emission proportional to the amount of analyte present to be generated shortly thereafter. 
     In  FIG. 2  the bound antibody-analyte complex  103  is captured in a very thin, well defined volume (normally some form of analysis slide, not shown) and presented at the sample plane  201 . Excitation light is generated by the light emitting diode (LED) source  208  then collimated by condenser system lenses  210 , filtered by the excitation filer  207 , shaped by the excitation aperture  206  and the projection lens  211 , redirected by a dichroic mirror  203 , and then passed through an objective lens  202  that acts to converge the excitation light rays down to an area appropriate for the very thin, well defined volume. The excitation system components contained in the dashed rectangle are called the excitation arm  209  of the optical detection system. Any captured and tagged analyte in that volume fluoresces, and a portion of that emission is intercepted by the objective lens  202 , passed through the dichroic mirror  203 , further passed through at least one band pass filter  204 , through a detector lens  212 , and finally through a detection aperture  213 . The emission light making it through the detection aperture  213  strikes the photodetector  205  and generates an electric current which is amplified into a usable signal. 
     At the photodetector  205 , the excitation arm  209  of the optical detection system delivers a photon flux (some number of photons per second) with some spectral distribution (i.e., some mix of wavelengths). This can be described by
 
Φ(χ)=φ· S (χ)  (1)
 
where φ is a scalar (units=photons/second) and S(χ) is a unitless shape function where the maximum value of S(χ) is unity. The magnitude of φ is determined by the output of the LED source  208 , the attenuation properties of the filters  207  and, the attenuation properties of the lenses  210 ,  211 , and  202 , the reflective properties of the dichroic mirror  203 , and the position tolerances of the optical elements. The characteristics of S(χ) are determined by the spectral properties of the LED source  208  and the transmission spectrum of the filter  207  and the reflective characteristics of the dichroic mirror  203 .
 
     If the NIST SRM 2944 glass is exposed to the photon flux Φ(χ) of equation (1), a composite emission curve will be obtained that can be approximated by summation over small increments of Δχ, i.e., the value of S(χ) at a particular χ times the normalized emission curve EC glass  at that wavelength. That is,
 
Σ X   S (χ)·ECglass(χ,ξ)  (2)
 
     The fluorescence photo flux Φ(ξ) emitted by the glass can be written as
 
Φ(χξ)=ψ·φ·Σ ξχ   S (ξχ)·Ecglass(χ,ξ)  (3)
 
where ψ is a scalar that is characteristic of the output of the NIST SRM 2944 glass.
 
     The electrical signal (current) that is generated at the detector at a particular emission wavelength χ can be described by
 
 E (ξ)=Φ(ξ)· G·S   R (ξ)  (4)
 
where G is a constant and S R (ξ) is a shape function such that the maximum value of S R (ξ) is unity. The magnitude of G is determined by the collection efficiency of the optics  202  and  212 , the transmission efficiency of the dichroic mirror  203  and emission filters  204 . The characteristics of S R (ξ) are determined by the spectral characteristics of the dichroic mirror  203 , emission filters  204  and the spectral characteristics of the detector (photodiode)  205 . The total electrical signal generated is
 
 E=Σ   ξ   G·S   R (ξ)=Φ(ξ)= G·Σ   ξ   S   R (ξ)·[ψ·φ·Σ χ   S (χ)·ECglass(χ,ξ)]  (5)
 
or,
 
 E=G·ψ·φ·Σ   ξ   S   R (ξ)·[Σ χ   S (χ)·ECglass(χ,ξ)]  (6)
 
     Suppose there is a master instrument “A” and a subordinate instrument “B” where Instrument “B” is to be normalized to instrument “A.” Using eqn. (6), the ratio of the signals (E A  and E B ) given by the two instruments in response to being presented with an identical NIST SRM 2944 glass target can be written as 
                         E   A       E   B       =       {         G   A     ·   ψ   ·     φ   A     ·       ∑   χ     ⁢         S   RA     ⁡     (   χ   )       ·     [       ∑   ξ     ⁢         S   A     ⁡     (   ξ   )       ·     ECglass   ⁡     (     ξ   ,   χ     )           ]               G   B     ·   ψ   ·     φ   B     ·       ∑   χ     ⁢         S   RB     ⁡     (   χ   )       ·     [       ∑   ξ     ⁢         S   B     ⁡     (   ξ   )       ·     ECglass   ⁡     (     ξ   ,   χ     )           ]             }     ⁢           ⁢   or       ,           (   7   )                 G   R     =       (         G   A     ·     φ   A           G   B     ·     φ   B         )     =       (       E   A       E   B       )     ·     {         ∑   χ     ⁢         S   RB     ⁡     (   χ   )       ·     [       ∑   ξ     ⁢         S   B     ⁡     (   ξ   )       ·     ECglass   ⁡     (     ξ   ,   χ     )           ]             ∑   χ     ⁢         S   RA     ⁡     (   χ   )       ·     [       ∑   ξ     ⁢         S   A     ⁡     (   ξ   )       ·     ECglass   ⁡     (     ξ   ,   χ     )           ]           }                 (   8   )               
where G R  is called the gain ratio. The responsivities of both instrument A and instrument B (S RA (ξ) and S RE (ξ), respectively) can be measured by presenting a constant intensity variable wavelength light source to each instrument in turn, sweeping the source through the range of wavelengths in the transmission band of the emission filters  204  and the dichroic mirror  203  while monitoring the signal generated by the respective instrument, then normalizing that signal by the maximum value obtained during that sweep. The emission spectra of both instruments, S A (χ) and S Z (χ), are easily measured by a spectrometer.
 
     Consider now the case of a fluorescent label, specifically Alexa Fluor® 647, where in  FIG. 4  the solid excitation curve  401  is designated by S DYE (χ). The equivalent digital values for S DYE (χ) are presented in Table 4. Also for Alexa Fluor® 647, in  FIG. 4  the dashed emission curve  402  is designated by S DYE (ξ). The equivalent digital values for S DYE (ξ) are presented in Table 5. 
     The photon flux Φ DYE (ξ) emitted by the fluorescent label (dye) can be written as
 
Φ DYE (ξ)=φ DYE ·φ·[Σ ξ   S (ξ)· S   DYE (ξ)]· S   DYE (χ)  (9)
 
where φ DYE  is a scalar that is characteristic of the output of the fluorescence label (dye).
 
     Rewriting eqn. (6) in terms of the fluorescence label (dye) gives 
     
       
         
           
             
               
                 
                   
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     Therefore, we can transform the response E B  from analyzer “B” to the response E A  that would be seen by the master analyzer “A” using eqn. (12). 
     This allows introduction of new fluorescence labels (dyes) to subordinate field instruments and allowing those subordinate field instruments to be re-normalized to a master analyzer “A” by simply providing the absorption and emission spectrum of the new fluorescence label (dye) and using eqn. (12). 
     In summary, the following is conducted in the factory for each subordinate instrument:
         Measure S Z (χ) and store this information on the instrument.   Measure S RZ (ξ)) and store this information on the instrument.   The Gain Ratio of each instrument is determined by scanning the NIST SRN 2944 calibration slide as a target, and then applying eqn. (8).       

     Exemplary Example of Factory Normalization 
     In this example, Analyzer AP106 is selected as the master instrument and Analyzer AP115 is selected as the subordinate instrument. The goal of this factory normalization is to determine the relationship between the two analyzers with respect to their individual responses to the same sample. This means that a response to a particular sample for the subordinate instrument can be converted to the response of the master instrument by multiplying the response of the subordinate instrument by the gain ratio and the remainder of the eqn. (12) to the right of G R  (as derived above and to be determined for this example below). The initial data gathering steps can be listed as follows:
         1. Obtain the normalized excitation intensity spectrum of AP106 (see Table 1 for the digital spectrophotometric data).   2. Obtain the normalized excitation intensity spectrum of AP115 (see Table 1 for the digital spectrophotometric data).   3. Obtain the normalized responsivity intensity spectrum of AP106 (see Table 2 for the digital spectrophotometric data).   4. Obtain the normalized responsivity intensity spectrum of AP115 (see Table 2 for the digital spectrophotometric data).   5. Obtain the normalized excitation/emission spectrum of NIST SRM 2944 glass (see Table 3A, 3B, and 3C for the digital spectrophotometric data).   6. Read the NIST SRM 2944 glass as a target in AP106 obtaining the response value for E A  of 2181.705 relative fluorescence units (RFU).   7. Read the NIST SRM 2944 glass as a target in AP115 obtaining the response value for E Z  of 2035.274 RFU.   8. Calculate the Gain ratio of AP106 to AP115 using eqn. (8) where the numerator in brackets has the value 123.9541 and the denominator in brackets has the value 126.4753 with a result as follows:       

     
       
         
           
             
               G 
               R 
             
             = 
             
               
                 
                   ( 
                   
                     2181.705 
                     2035.274 
                   
                   ) 
                 
                 * 
                 
                   ( 
                   
                     123.9541 
                     126.4763 
                   
                   ) 
                 
               
               = 
               1.0506 
             
           
         
       
         
         
           
              Note that the ratio (123.9541/126.4763) expresses the difference in signal between the two instruments based on spectral differences. Whereas the gain ratio G R  expresses differences due to non-spectral differences (e.g., one instrument may have a slightly brighter illumination LED or somewhat more efficient receiver optics). 
             9. Obtain the relative absorption spectrum of the fluorescent label (dye) used in the analysis. In this case Alexa Fluor® 647 is being used and the associated digital spectrophotometric data is presented in Table 4. 
             10. Obtain the relative emission intensity of the fluorescent label (dye) used in the analysis. In this case Alexa Fluor® 647 is being used and the associated digital spectrophotometric data is presented in Table 5. 
             11. Using eqn. (12), the normalization factor between AP115 responses and AP106 responses can be determined as follows where the numerator in eqn. (12) is 122.1005 and the denominator in eqn. (12) is 117.6860: 
           
         
       
    
     
       
         
           
             
               
                 E 
                 A 
               
               
                 E 
                 Z 
               
             
             = 
             
               
                 
                   ( 
                   1.0506 
                   ) 
                 
                 * 
                 
                   ( 
                   
                     117.6860 
                     122.1005 
                   
                   ) 
                 
               
               = 
               1.0126 
             
           
         
       
         
         
           
              That is, to convert a response from AP115 to a response normalized to AP106 when making measurements with Alexa Fluor® 647, we must multiply the AP115 responses by 1.0126. 
           
         
       
    
     Exemplary Example of Field Re-Normalization 
     In this example, subordinate instrument AP115 has been previously normalized to master instrument AP106 and it is desired to introduce a new fluorescent label (dye). The prior analysis allows introduction of new fluorescent labels (dyes) to subordinate field instruments and allowing those subordinate field instruments to normalize to the master analyzer by simply providing the absorption and emission spectrum of the new dye and using eqn. (12) above. The method is outlined as follows:
         1. Obtain the relative absorption spectrum of the fluorescent label (dye) used in the analysis. In this case Alexa Fluor® 635 is being used, see  FIG. 3 , solid excitation curve  301  and the associated digital spectrophotometric data is presented in Table 6.   2. Obtain the relative emission intensity of the fluorescent label (dye) used in the analysis. In this case Alexa Fluor® 635 is being used, see  FIG. 3 , dashed emission curve  302  and the associated digital spectrophotometric data is presented in Table 7.   3. Using eqn. (12), the normalization factor between AP115 responses and AP106 responses can be determined as follows where the numerator in eqn. (12) is 44.02245 and the denominator in eqn. (12) is 45.2193:       

     
       
         
           
             
               
                 E 
                 A 
               
               
                 E 
                 Z 
               
             
             = 
             
               
                 
                   ( 
                   1.0506 
                   ) 
                 
                 * 
                 
                   ( 
                   
                     44.02245 
                     45.2194 
                   
                   ) 
                 
               
               = 
               1.0274 
             
           
         
       
         
         
           
              That is, to convert a signal from AP115 to a signal normalized to AP106 when making measurements with Alexa Fluor® 635 as opposed to Alexa Fluor® 647, we must multiply the AP115 signals by 1.01274. Note that the Gain Ratio is not dependent upon the fluorescent label (dye) and remains constant. 
           
         
       
    
     In practice, when a new fluorescence label (dye) is introduced to a subordinate field instrument, the quantity
 
Σ χ   S   RB (ξ)·[Σ ξχ   S   B (χ)· S   DYE (χ)]· S   DYE (ξ)
         will be provided to that instrument along with
 
 S   DYE (ξ) and  S   DYE (χ)
   so that it is capable of running assays that make use of that new fluorescence label (dye).       

     Exemplary Example of Factory Linear Calibration 
     In this example, a standard calibration procedure will be conducted with samples or specimens of known analyte concentration. The procedure will utilize 10 samples having known analyte concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ng/mL. The master instrument responses were observed to be 1.15, 1.90, 3.10, 3.90, 5.05, 5.95, 7.30, 7.90, 8.90, and 10.20. For fluorometry, the amount of emitted light is frequently proportional to amount of analyte present in the sample or specimen; hence, a linear calibration curve is usually employed. Here the known concentration values, which are known without error, are used as the predictor variable and the master instrument responses, containing measurement error, are used as the response variable. This situation is known to satisfy the requirement for using ordinary least squares (OLS) regression. The above data and the fitted regression line are shown in  FIG. 5 . The data points  501  and the fitted OLS regression line (shown as a solid black line)  502  indicate a close correlation for the master instrument. 
     For the master instrument, the instrument response to a sample or specimen (as indicated by a y-axis value) is traced back to the right horizontally to the fitted linear calibration line and then traced vertically to the x-axis to obtain the estimate of the analyte concentration in the sample or specimen. For example, in  FIG. 5 , if the master instrument response to a sample or specimen is 5, then tracing to the right, the horizontal line intercepts the linear calibration line and tracing down a value of approximately 5 on the x-axis is obtained. 
     For the subordinate instruments, subordinate instrument response to a sample or specimen is multiplied by the Gain Ratio and everything to the right of E Z  in eqn. (1) such that the resulting response can be used just as if it were obtained from the master instrument to obtain an estimate of the analyte concentration in the sample or specimen. For this specific case, the subordinate instrument would produce a response of 4 and subsequently that response would be multiplied by the gain factor of 1.25 to yield an equivalent master instrument response of 5. Also using the master calibration curve produces an estimated analyte concentration of approximately 5. 
     Exemplary Example of Field Linear Re-Calibration 
     In this example, a new fluorescent label (dye) has been introduced into the analysis chemistry and the subordinate instruments in the field have been re-normalized. For this situation there are two methods that can be used to obtain appropriate estimates of the analyte concentration in the sample or specimen as follows:
         1. The subordinate instrument response can be multiplied by the normalization factor and the old factory calibration curve can be used. In  FIG. 5 , for a subordinate instrument response of 4 and a normalization factor of 1.25, this is represented by the up arrow indicating that 4*1.25=5 is the equivalent master instrument response. Starting a 5 on the y-axis and tracing right to the master linear calibration line  502  and then down to the x-axis produces an estimate of 5.   2. Alternatively, a new linear calibration  503  curve can be constructed by multiplying the slope of the old linear calibration curve  502  by the inverse of the normalization factor. The new calibration curve  503  would then, as in in  FIG. 5 , have a slope of 1.005*(1/1.25)=0.804. The estimate of the analyte concentration in the sample or specimen would then be obtained by starting on the y-axis at the subordinate instrument response (4) then tracing to the right until the new linear calibration curve is encountered and then moving down to the x-axis. It is clear from  FIG. 5  that this procedure or the procedure 1 above that both result in the same analyte concentration estimates.       

     Exemplary Example of Factory Non-Linear Calibration 
     In this example, the amount of emitted light is not proportional to amount of analyte present in the sample or specimen; hence, a linear calibration curve cannot be used. In a manner similar to the linear calibration example, a series of 10 samples or specimens of known analyte concentrations is prepared and a non-linear sigmoidal function  601  is fitted to the data points  602 . Hence, for the master instrument, a y-axis response is converted into an estimate of analyte concentration, by starting at the y-axis value, tracing right until the calibration curve is encountered and then tracing down to the x-axis to obtain the analyte concentration estimate. 
     Exemplary Example of Field Non-Linear Re-Calibration 
     In this example,  FIG. 6  shows a non-linear (s-shaped) calibration curve  601 . Assuming that the normalization factor is 1.25 between the master and subordinate instruments, a subordinate instrument response of 4 would generate an equivalent master instrument response of (4*1.25)=5 which would produce an estimated analyte concentration of approximately 5½. Unlike the linear calibration case, the slope of the non-linear calibration curve cannot be easily adjusted such that the raw subordinate instrument response can be used. Here it is necessary to multiply the subordinate instrument response by the normalization factor to obtain the equivalent master instrument response and then use the master instrument calibration curve. 
     Simulation Test of Normalization Factor Effectiveness 
     To test the effectiveness of the normalization process, an initial Monte Carlo simulation was conducted where 10,000 simulated fluorescence analyzers were presented with a fixed amount of Alexa Fluor® 647 (AF 647) fluorescence label (dye) or a fixed amount of bismuth-doped glass as used in the NIST SRM 2944 standard. Sources of variation were as follows:
         1. Incubation temperature was allowed to vary between 36° C. and 38° C.   2. The bandpass characteristics of 3 optical filters in the detection arm of the optical detection system were allowed to vary (according the manufacturers specification)   3. The excitation wavelength was allowed to vary between 630 nm and 636 nm.       

     After 10,000 simulated analyzers were configured according to the above, the model generated the statistics of a variety of responses.  FIG. 7  shows the resulting histogram  701  of responses to simulated samples using AF 647 dye. There is about a +/−25% range in the population response.  FIG. 8  shows the population of ratios of each individual analyzer&#39;s response to AF 647 and the NIST SRM 2944 standard. The resulting histogram of responses  801  shows a spread of about +/−20%. Clearly, the raw response of the NIST SRM 2944 does a poor job in predicting the response of the instrument to the samples using AF 647. However, if the same analysis is repeated where a normalization factor is employed and measurement noise (error) in the following is allowed:
         1. Noise associated with the analyzer aligning and reading the calibration slide. A value was used of 0.5% CV that should be achievable with 4 load and align events each with 4 reads of the fluorescent label (dye). The alignment process was actually a fairly significant source of variability.   2. Noise associated with the characterization of the analyzer&#39;s excitation and responsivity spectra by factory calibration instrumentation. It was estimated that each data point had a 0.25% CV imprecision.   3. Noise associated with the characterization of the analyzer&#39;s incubation temperature. Noise of one standard deviation was taken to be 0.067° C. This creates an error because of the temperature sensitivity differences between the NIST SRM 2944 glass (−0.25% per ° C.) and the AF 647 fluorescent label (dye) (−1.2% per ° C.).       

       FIG. 9  contains the resulting histogram of errors  901  which indicates that the overall analyte estimation error has been reduced to about a range of +/−1%. 
     It will be apparent to those skilled in the art that various modifications and variations can be made to the article of manufacture disclosed herein. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents. 
     The disclosure of all publications cited above is expressly incorporated herein by reference in their entireties to the same extent as if each were incorporated by reference individually. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Normalized Excitation Intensity of Analyzers AP106 and AP115 
               
            
           
           
               
               
               
            
               
                 Wavelength 
                 Normalized Excitation 
                 Normalized Excitation 
               
               
                 (nm) 
                 Intensity AP106 
                 Intensity AP115 
               
               
                   
               
            
           
           
               
               
               
            
               
                 610 
                 0.002 
                 0.001 
               
               
                 611 
                 0.001 
                 0 
               
               
                 612 
                 0 
                 0.002 
               
               
                 613 
                 0 
                 0.001 
               
               
                 614 
                 0 
                 0.002 
               
               
                 615 
                 0.002 
                 0.002 
               
               
                 616 
                 0.002 
                 0.003 
               
               
                 617 
                 0.002 
                 0.002 
               
               
                 618 
                 0.001 
                 0.002 
               
               
                 619 
                 0.002 
                 0.004 
               
               
                 620 
                 0.002 
                 0.006 
               
               
                 621 
                 0.002 
                 0.003 
               
               
                 622 
                 0.003 
                 0.006 
               
               
                 623 
                 0.002 
                 0.003 
               
               
                 624 
                 0.002 
                 0.006 
               
               
                 625 
                 0.003 
                 0.004 
               
               
                 626 
                 0.003 
                 0.004 
               
               
                 627 
                 0.003 
                 0.008 
               
               
                 628 
                 0.004 
                 0.008 
               
               
                 629 
                 0.005 
                 0.01 
               
               
                 630 
                 0.007 
                 0.01 
               
               
                 631 
                 0.018 
                 0.015 
               
               
                 632 
                 0.107 
                 0.08 
               
               
                 633 
                 0.749 
                 0.588 
               
               
                 634 
                 1 
                 1 
               
               
                 635 
                 1 
                 1 
               
               
                 636 
                 0.902 
                 0.888 
               
               
                 637 
                 0.783 
                 0.776 
               
               
                 638 
                 0.649 
                 0.664 
               
               
                 639 
                 0.531 
                 0.547 
               
               
                 640 
                 0.422 
                 0.433 
               
               
                 641 
                 0.33 
                 0.339 
               
               
                 642 
                 0.264 
                 0.287 
               
               
                 643 
                 0.201 
                 0.229 
               
               
                 644 
                 0.17 
                 0.195 
               
               
                 645 
                 0.138 
                 0.169 
               
               
                 646 
                 0.106 
                 0.134 
               
               
                 647 
                 0.089 
                 0.114 
               
               
                 648 
                 0.071 
                 0.094 
               
               
                 649 
                 0.052 
                 0.077 
               
               
                 650 
                 0.018 
                 0.033 
               
               
                 651 
                 0.011 
                 0.023 
               
               
                 652 
                 0.014 
                 0.025 
               
               
                 653 
                 0.015 
                 0.026 
               
               
                 654 
                 0.013 
                 0.026 
               
               
                 655 
                 0.016 
                 0.026 
               
               
                 656 
                 0.016 
                 0.028 
               
               
                 657 
                 0.017 
                 0.029 
               
               
                 658 
                 0.016 
                 0.028 
               
               
                 659 
                 0.019 
                 0.029 
               
               
                 660 
                 0.02 
                 0.033 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Normalized Responsivity of Analyzers AP106 and AP115 
               
            
           
           
               
               
               
            
               
                 Wavelength 
                 Normalized 
                 Normalized 
               
               
                 (nm) 
                 Responsivity AP106 
                 Responsivity AP115 
               
               
                   
               
            
           
           
               
               
               
            
               
                 660 
                 0.000 
                 0 
               
               
                 661 
                 0.000 
                 0 
               
               
                 662 
                 0.000 
                 0 
               
               
                 663 
                 0.000 
                 0 
               
               
                 664 
                 0.000 
                 0 
               
               
                 665 
                 0.001 
                 0.003 
               
               
                 666 
                 0.007 
                 0.033 
               
               
                 667 
                 0.076 
                 0.169 
               
               
                 668 
                 0.296 
                 0.503 
               
               
                 669 
                 0.721 
                 0.857 
               
               
                 670 
                 0.931 
                 0.944 
               
               
                 671 
                 0.963 
                 0.963 
               
               
                 672 
                 0.976 
                 0.974 
               
               
                 673 
                 0.984 
                 0.981 
               
               
                 674 
                 0.987 
                 0.986 
               
               
                 675 
                 0.989 
                 0.987 
               
               
                 676 
                 0.987 
                 0.988 
               
               
                 677 
                 0.989 
                 0.99 
               
               
                 678 
                 0.990 
                 0.991 
               
               
                 679 
                 0.990 
                 0.992 
               
               
                 680 
                 0.992 
                 0.995 
               
               
                 681 
                 0.996 
                 0.999 
               
               
                 682 
                 0.999 
                 1 
               
               
                 683 
                 1.000 
                 0.999 
               
               
                 684 
                 1.000 
                 0.998 
               
               
                 685 
                 0.998 
                 0.997 
               
               
                 686 
                 0.997 
                 0.996 
               
               
                 687 
                 0.995 
                 0.995 
               
               
                 688 
                 0.994 
                 0.991 
               
               
                 689 
                 0.991 
                 0.977 
               
               
                 690 
                 0.973 
                 0.942 
               
               
                 691 
                 0.932 
                 0.886 
               
               
                 692 
                 0.874 
                 0.825 
               
               
                 693 
                 0.813 
                 0.758 
               
               
                 694 
                 0.747 
                 0.677 
               
               
                 695 
                 0.661 
                 0.577 
               
               
                 696 
                 0.557 
                 0.494 
               
               
                 697 
                 0.476 
                 0.444 
               
               
                 698 
                 0.431 
                 0.361 
               
               
                 699 
                 0.348 
                 0.186 
               
               
                 700 
                 0.147 
                 0.039 
               
               
                 701 
                 0.028 
                 0.007 
               
               
                 702 
                 0.006 
                 0.002 
               
               
                 703 
                 0.002 
                 0.001 
               
               
                 704 
                 0.001 
                 0 
               
               
                 705 
                 0.000 
                 0 
               
               
                 706 
                 0.000 
                 0 
               
               
                 707 
                 0.000 
                 0 
               
               
                 708 
                 0.000 
                 0 
               
               
                 709 
                 0.000 
                 0 
               
               
                 710 
                 0.000 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3A 
               
             
            
               
                   
               
               
                 Normalized Excitation/Emission Spectrum of NIST SRM 2944 Glass 
               
               
                 (Excitation Range of 630 nm to 638 nm) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
               
               
                 Emission λ 
                 630 nm 
                 631 nm 
                 632 nm 
                 633 nm 
                 634 nm 
                 635 nm 
                 636 nm 
                 637 nm 
                 638 nm 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 660 nm 
                 0.34549 
                 0.33956 
                 0.33023 
                 0.32210 
                 0.31582 
                 0.30802 
                 0.29942 
                 0.29206 
                 0.28678 
               
               
                 661 nm 
                 0.35772 
                 0.34993 
                 0.34102 
                 0.33521 
                 0.32440 
                 0.31786 
                 0.31031 
                 0.30331 
                 0.29702 
               
               
                 662 nm 
                 0.36896 
                 0.36160 
                 0.35298 
                 0.34501 
                 0.33518 
                 0.33052 
                 0.32255 
                 0.31517 
                 0.30726 
               
               
                 663 nm 
                 0.38392 
                 0.37425 
                 0.36760 
                 0.35853 
                 0.34984 
                 0.34257 
                 0.33388 
                 0.32481 
                 0.31928 
               
               
                 664 nm 
                 0.39699 
                 0.39019 
                 0.37935 
                 0.37124 
                 0.36228 
                 0.35438 
                 0.34674 
                 0.33794 
                 0.33082 
               
               
                 665 nm 
                 0.41212 
                 0.40123 
                 0.39331 
                 0.38280 
                 0.37668 
                 0.36648 
                 0.35845 
                 0.35121 
                 0.34225 
               
               
                 666 nm 
                 0.42312 
                 0.41395 
                 0.40579 
                 0.39620 
                 0.38547 
                 0.37763 
                 0.36996 
                 0.36349 
                 0.35335 
               
               
                 667 nm 
                 0.43856 
                 0.42995 
                 0.41957 
                 0.41035 
                 0.40103 
                 0.39396 
                 0.38782 
                 0.37574 
                 0.36701 
               
               
                 668 nm 
                 0.45547 
                 0.44361 
                 0.43619 
                 0.42548 
                 0.41737 
                 0.40622 
                 0.39897 
                 0.39043 
                 0.38220 
               
               
                 669 nm 
                 0.46686 
                 0.45881 
                 0.45030 
                 0.44020 
                 0.43125 
                 0.42149 
                 0.41088 
                 0.40334 
                 0.39448 
               
               
                 670 nm 
                 0.48376 
                 0.47254 
                 0.46310 
                 0.45249 
                 0.44417 
                 0.43516 
                 0.42428 
                 0.41614 
                 0.40696 
               
               
                 671 nm 
                 0.49900 
                 0.48850 
                 0.47782 
                 0.46725 
                 0.46050 
                 0.44937 
                 0.43894 
                 0.43078 
                 0.42308 
               
               
                 672 nm 
                 0.51526 
                 0.50339 
                 0.49342 
                 0.48204 
                 0.47241 
                 0.46424 
                 0.45153 
                 0.44363 
                 0.43545 
               
               
                 673 nm 
                 0.52976 
                 0.51942 
                 0.50948 
                 0.49668 
                 0.48821 
                 0.47739 
                 0.46932 
                 0.45703 
                 0.44964 
               
               
                 674 nm 
                 0.54523 
                 0.53238 
                 0.52233 
                 0.51327 
                 0.50100 
                 0.49177 
                 0.48550 
                 0.47438 
                 0.46410 
               
               
                 675 nm 
                 0.56020 
                 0.55060 
                 0.53721 
                 0.52785 
                 0.51904 
                 0.50735 
                 0.49493 
                 0.48674 
                 0.47747 
               
               
                 676 nm 
                 0.57498 
                 0.56199 
                 0.55172 
                 0.53999 
                 0.53040 
                 0.51979 
                 0.51242 
                 0.50051 
                 0.49118 
               
               
                 677 nm 
                 0.58950 
                 0.57750 
                 0.56461 
                 0.55440 
                 0.54368 
                 0.53247 
                 0.52302 
                 0.51226 
                 0.50388 
               
               
                 678 nm 
                 0.60211 
                 0.58952 
                 0.57800 
                 0.56891 
                 0.55852 
                 0.54858 
                 0.53826 
                 0.52581 
                 0.51567 
               
               
                 679 nm 
                 0.61899 
                 0.60799 
                 0.59682 
                 0.58456 
                 0.57233 
                 0.56236 
                 0.55323 
                 0.54233 
                 0.53079 
               
               
                 680 nm 
                 0.63571 
                 0.62416 
                 0.61074 
                 0.59914 
                 0.59201 
                 0.57863 
                 0.56691 
                 0.55660 
                 0.54640 
               
               
                 681 nm 
                 0.65119 
                 0.63768 
                 0.62549 
                 0.61473 
                 0.60436 
                 0.59340 
                 0.58563 
                 0.57149 
                 0.55993 
               
               
                 682 nm 
                 0.66555 
                 0.65240 
                 0.64012 
                 0.63216 
                 0.62131 
                 0.60897 
                 0.59902 
                 0.58693 
                 0.57436 
               
               
                 683 nm 
                 0.68193 
                 0.66992 
                 0.65779 
                 0.64809 
                 0.63722 
                 0.62455 
                 0.61273 
                 0.60073 
                 0.59146 
               
               
                 684 nm 
                 0.69631 
                 0.68391 
                 0.67507 
                 0.66524 
                 0.65241 
                 0.63948 
                 0.62716 
                 0.61849 
                 0.60849 
               
               
                 685 nm 
                 0.71191 
                 0.69806 
                 0.68725 
                 0.67714 
                 0.66511 
                 0.65241 
                 0.64263 
                 0.63119 
                 0.62026 
               
               
                 686 nm 
                 0.72485 
                 0.71479 
                 0.70406 
                 0.68995 
                 0.68133 
                 0.66728 
                 0.65735 
                 0.64566 
                 0.63500 
               
               
                 687 nm 
                 0.74255 
                 0.72878 
                 0.71670 
                 0.70500 
                 0.69137 
                 0.68162 
                 0.67083 
                 0.65914 
                 0.64813 
               
               
                 688 nm 
                 0.75576 
                 0.74375 
                 0.73268 
                 0.72053 
                 0.70923 
                 0.69377 
                 0.68607 
                 0.67391 
                 0.66217 
               
               
                 689 nm 
                 0.76864 
                 0.75885 
                 0.74478 
                 0.73313 
                 0.72375 
                 0.70988 
                 0.69743 
                 0.68662 
                 0.67722 
               
               
                 690 nm 
                 0.78382 
                 0.76997 
                 0.76045 
                 0.74728 
                 0.73515 
                 0.72335 
                 0.70914 
                 0.69861 
                 0.68906 
               
               
                 691 nm 
                 0.79956 
                 0.78754 
                 0.77465 
                 0.76029 
                 0.75030 
                 0.73720 
                 0.72440 
                 0.71331 
                 0.70199 
               
               
                 692 nm 
                 0.81396 
                 0.80383 
                 0.78749 
                 0.77734 
                 0.76329 
                 0.75653 
                 0.74207 
                 0.72610 
                 0.71928 
               
               
                 693 nm 
                 0.83057 
                 0.81751 
                 0.80235 
                 0.79029 
                 0.77913 
                 0.76468 
                 0.75417 
                 0.74559 
                 0.73236 
               
               
                 694 nm 
                 0.84067 
                 0.83041 
                 0.81674 
                 0.80409 
                 0.78959 
                 0.77977 
                 0.77171 
                 0.75430 
                 0.74347 
               
               
                 695 nm 
                 0.85438 
                 0.84491 
                 0.83224 
                 0.81948 
                 0.80731 
                 0.79462 
                 0.78425 
                 0.77103 
                 0.76048 
               
               
                 696 nm 
                 0.87009 
                 0.85938 
                 0.84873 
                 0.83082 
                 0.81966 
                 0.80737 
                 0.79485 
                 0.78336 
                 0.77542 
               
               
                 697 nm 
                 0.88250 
                 0.87089 
                 0.85528 
                 0.84315 
                 0.83042 
                 0.82140 
                 0.80550 
                 0.79784 
                 0.78385 
               
               
                 698 nm 
                 0.89514 
                 0.88045 
                 0.86759 
                 0.85686 
                 0.84831 
                 0.83358 
                 0.82316 
                 0.81013 
                 0.79985 
               
               
                 699 nm 
                 0.90298 
                 0.89312 
                 0.87890 
                 0.86638 
                 0.85667 
                 0.84326 
                 0.82821 
                 0.82216 
                 0.80856 
               
               
                 700 nm 
                 0.91588 
                 0.90337 
                 0.88566 
                 0.87942 
                 0.86508 
                 0.85438 
                 0.84473 
                 0.83000 
                 0.81662 
               
               
                 701 nm 
                 0.92416 
                 0.91464 
                 0.90406 
                 0.88702 
                 0.87977 
                 0.86353 
                 0.85174 
                 0.84086 
                 0.83206 
               
               
                 702 nm 
                 0.93556 
                 0.92286 
                 0.91033 
                 0.90265 
                 0.88861 
                 0.87638 
                 0.86469 
                 0.85091 
                 0.83958 
               
               
                 703 nm 
                 0.94464 
                 0.92976 
                 0.92086 
                 0.90517 
                 0.89383 
                 0.88544 
                 0.86948 
                 0.86311 
                 0.84956 
               
               
                 704 nm 
                 0.95172 
                 0.94134 
                 0.92452 
                 0.91652 
                 0.90713 
                 0.89536 
                 0.87968 
                 0.86897 
                 0.85622 
               
               
                 705 nm 
                 0.96445 
                 0.94477 
                 0.94112 
                 0.92292 
                 0.91835 
                 0.90046 
                 0.88922 
                 0.87454 
                 0.86843 
               
               
                 706 nm 
                 0.97045 
                 0.95788 
                 0.95020 
                 0.93519 
                 0.92430 
                 0.91298 
                 0.89947 
                 0.88786 
                 0.87275 
               
               
                 707 nm 
                 0.98165 
                 0.96576 
                 0.95652 
                 0.94251 
                 0.93266 
                 0.92085 
                 0.90612 
                 0.89648 
                 0.88468 
               
               
                 708 nm 
                 0.98706 
                 0.97196 
                 0.96089 
                 0.95344 
                 0.93654 
                 0.92808 
                 0.91656 
                 0.90630 
                 0.89525 
               
               
                 709 nm 
                 0.99556 
                 0.97889 
                 0.96765 
                 0.95331 
                 0.94638 
                 0.93428 
                 0.91999 
                 0.91175 
                 0.90195 
               
               
                  10 nm 
                 1.00000 
                 0.98436 
                 0.97267 
                 0.95728 
                 0.95293 
                 0.93689 
                 0.92688 
                 0.91517 
                 0.90614 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3B 
               
             
            
               
                   
               
               
                 Normalized Excitation/Emission Spectrum of NIST SRM 2944 Glass 
               
               
                 (Excitation Range of 639 nm to 647 nm) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
               
               
                 Emission λ 
                 639 nm 
                 640 nm 
                 641 nm 
                 642 nm 
                 643 nm 
                 644 nm 
                 645 nm 
                 646 nm 
                 647 nm 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 660 nm 
                 0.27911 
                 0.27185 
                 0.26452 
                 0.25901 
                 0.25135 
                 0.24785 
                 0.23902 
                 0.23594 
                 0.23167 
               
               
                 661 nm 
                 0.28995 
                 0.28094 
                 0.27595 
                 0.26975 
                 0.26183 
                 0.25660 
                 0.25034 
                 0.24434 
                 0.23738 
               
               
                 662 nm 
                 0.30021 
                 0.29278 
                 0.28366 
                 0.27893 
                 0.27137 
                 0.26506 
                 0.25849 
                 0.25475 
                 0.24551 
               
               
                 663 nm 
                 0.31158 
                 0.30306 
                 0.29624 
                 0.28923 
                 0.28192 
                 0.27477 
                 0.26761 
                 0.26381 
                 0.25771 
               
               
                 664 nm 
                 0.32579 
                 0.31660 
                 0.30891 
                 0.30162 
                 0.29329 
                 0.28622 
                 0.27996 
                 0.27318 
                 0.26751 
               
               
                 665 nm 
                 0.33523 
                 0.32804 
                 0.31951 
                 0.31145 
                 0.30404 
                 0.29706 
                 0.28971 
                 0.28405 
                 0.27674 
               
               
                 666 nm 
                 0.34443 
                 0.33741 
                 0.33095 
                 0.32289 
                 0.31555 
                 0.30954 
                 0.30037 
                 0.29415 
                 0.28606 
               
               
                 667 nm 
                 0.35897 
                 0.35202 
                 0.34426 
                 0.33462 
                 0.32785 
                 0.32013 
                 0.31137 
                 0.30462 
                 0.29813 
               
               
                 668 nm 
                 0.37347 
                 0.36475 
                 0.35725 
                 0.34954 
                 0.34101 
                 0.33435 
                 0.32413 
                 0.31842 
                 0.30883 
               
               
                 669 nm 
                 0.38590 
                 0.37578 
                 0.36848 
                 0.36081 
                 0.35423 
                 0.34506 
                 0.33669 
                 0.33021 
                 0.31995 
               
               
                 670 nm 
                 0.39942 
                 0.38957 
                 0.38234 
                 0.37419 
                 0.36530 
                 0.35769 
                 0.34828 
                 0.34083 
                 0.33246 
               
               
                 671 nm 
                 0.41226 
                 0.40227 
                 0.39425 
                 0.38722 
                 0.37700 
                 0.37099 
                 0.35937 
                 0.35261 
                 0.34617 
               
               
                 672 nm 
                 0.42698 
                 0.41667 
                 0.40787 
                 0.39921 
                 0.39076 
                 0.38205 
                 0.37587 
                 0.36751 
                 0.36019 
               
               
                 673 nm 
                 0.43981 
                 0.43263 
                 0.42078 
                 0.41488 
                 0.40511 
                 0.39550 
                 0.38413 
                 0.37933 
                 0.36968 
               
               
                 674 nm 
                 0.45412 
                 0.44475 
                 0.43423 
                 0.42555 
                 0.41891 
                 0.40664 
                 0.40179 
                 0.39040 
                 0.38156 
               
               
                 675 nm 
                 0.46688 
                 0.45568 
                 0.44866 
                 0.43969 
                 0.43155 
                 0.42118 
                 0.41290 
                 0.40209 
                 0.39604 
               
               
                 676 nm 
                 0.48073 
                 0.47073 
                 0.46036 
                 0.45261 
                 0.44345 
                 0.43448 
                 0.42458 
                 0.41554 
                 0.40821 
               
               
                 677 nm 
                 0.49339 
                 0.48261 
                 0.47440 
                 0.46501 
                 0.45556 
                 0.44665 
                 0.43779 
                 0.42705 
                 0.41914 
               
               
                 678 nm 
                 0.50585 
                 0.49479 
                 0.48616 
                 0.47395 
                 0.46894 
                 0.45722 
                 0.44763 
                 0.43920 
                 0.43098 
               
               
                 679 nm 
                 0.51982 
                 0.51266 
                 0.50154 
                 0.49310 
                 0.48233 
                 0.47278 
                 0.46128 
                 0.45399 
                 0.44395 
               
               
                 680 nm 
                 0.53750 
                 0.52403 
                 0.51516 
                 0.50484 
                 0.49664 
                 0.48659 
                 0.47725 
                 0.46932 
                 0.45799 
               
               
                 681 nm 
                 0.54993 
                 0.54309 
                 0.52722 
                 0.51938 
                 0.50822 
                 0.50052 
                 0.49061 
                 0.48152 
                 0.47112 
               
               
                 682 nm 
                 0.56629 
                 0.55587 
                 0.54475 
                 0.53432 
                 0.52582 
                 0.51478 
                 0.50473 
                 0.49179 
                 0.48528 
               
               
                 683 nm 
                 0.58150 
                 0.56910 
                 0.55823 
                 0.55040 
                 0.54175 
                 0.53082 
                 0.51382 
                 0.50954 
                 0.49833 
               
               
                 684 nm 
                 0.59626 
                 0.58549 
                 0.57161 
                 0.56348 
                 0.55152 
                 0.54292 
                 0.53166 
                 0.52173 
                 0.51299 
               
               
                 685 nm 
                 0.60793 
                 0.59802 
                 0.58731 
                 0.57827 
                 0.56494 
                 0.55536 
                 0.54588 
                 0.53733 
                 0.52397 
               
               
                 686 nm 
                 0.62306 
                 0.61432 
                 0.59987 
                 0.58847 
                 0.58125 
                 0.57004 
                 0.55623 
                 0.54756 
                 0.53980 
               
               
                 687 nm 
                 0.63897 
                 0.62686 
                 0.61389 
                 0.60637 
                 0.59225 
                 0.58169 
                 0.57082 
                 0.56042 
                 0.55440 
               
               
                 688 nm 
                 0.65070 
                 0.64051 
                 0.62660 
                 0.61937 
                 0.60813 
                 0.59850 
                 0.58688 
                 0.57622 
                 0.56656 
               
               
                 689 nm 
                 0.66528 
                 0.65593 
                 0.64145 
                 0.63173 
                 0.62156 
                 0.60885 
                 0.59913 
                 0.58936 
                 0.57848 
               
               
                 690 nm 
                 0.67591 
                 0.66659 
                 0.65579 
                 0.64355 
                 0.63498 
                 0.62411 
                 0.61001 
                 0.60088 
                 0.59155 
               
               
                 691 nm 
                 0.69007 
                 0.67921 
                 0.67153 
                 0.65932 
                 0.64955 
                 0.63466 
                 0.62460 
                 0.61504 
                 0.60294 
               
               
                 692 nm 
                 0.70667 
                 0.69526 
                 0.68166 
                 0.67403 
                 0.66398 
                 0.65158 
                 0.63974 
                 0.62803 
                 0.61911 
               
               
                 693 nm 
                 0.72124 
                 0.71108 
                 0.69785 
                 0.68725 
                 0.67435 
                 0.66574 
                 0.65178 
                 0.64059 
                 0.63174 
               
               
                 694 nm 
                 0.73618 
                 0.72511 
                 0.71025 
                 0.69935 
                 0.69291 
                 0.68093 
                 0.66894 
                 0.65427 
                 0.64630 
               
               
                 695 nm 
                 0.74781 
                 0.73408 
                 0.72458 
                 0.71131 
                 0.70287 
                 0.69274 
                 0.68404 
                 0.67117 
                 0.65988 
               
               
                 696 nm 
                 0.76197 
                 0.74705 
                 0.73821 
                 0.72851 
                 0.71581 
                 0.70501 
                 0.69595 
                 0.68463 
                 0.67072 
               
               
                 697 nm 
                 0.77559 
                 0.76219 
                 0.75201 
                 0.74229 
                 0.72922 
                 0.71381 
                 0.70528 
                 0.69469 
                 0.68236 
               
               
                 698 nm 
                 0.78480 
                 0.77821 
                 0.76451 
                 0.75169 
                 0.73934 
                 0.73148 
                 0.71671 
                 0.70809 
                 0.69831 
               
               
                 699 nm 
                 0.79627 
                 0.78192 
                 0.77271 
                 0.76445 
                 0.74887 
                 0.74191 
                 0.73119 
                 0.71864 
                 0.70551 
               
               
                 700 nm 
                 0.80875 
                 0.79373 
                 0.78544 
                 0.77658 
                 0.75904 
                 0.75085 
                 0.73887 
                 0.72835 
                 0.71694 
               
               
                 701 nm 
                 0.82209 
                 0.80501 
                 0.79439 
                 0.78660 
                 0.77305 
                 0.75991 
                 0.75274 
                 0.74043 
                 0.73168 
               
               
                 702 nm 
                 0.82774 
                 0.81885 
                 0.80382 
                 0.79314 
                 0.78494 
                 0.76795 
                 0.76162 
                 0.75244 
                 0.73873 
               
               
                 703 nm 
                 0.83419 
                 0.82483 
                 0.81362 
                 0.80471 
                 0.79361 
                 0.78072 
                 0.77200 
                 0.75994 
                 0.74859 
               
               
                 704 nm 
                 0.85007 
                 0.83739 
                 0.82417 
                 0.81290 
                 0.80216 
                 0.78760 
                 0.77658 
                 0.77141 
                 0.75671 
               
               
                 705 nm 
                 0.85479 
                 0.84240 
                 0.83616 
                 0.82402 
                 0.80819 
                 0.79908 
                 0.78787 
                 0.77910 
                 0.76791 
               
               
                 706 nm 
                 0.86330 
                 0.85349 
                 0.84185 
                 0.82907 
                 0.81821 
                 0.80705 
                 0.79720 
                 0.79097 
                 0.77680 
               
               
                 707 nm 
                 0.87533 
                 0.85838 
                 0.85322 
                 0.84090 
                 0.82425 
                 0.82040 
                 0.80514 
                 0.79461 
                 0.78601 
               
               
                 708 nm 
                 0.87902 
                 0.87176 
                 0.85697 
                 0.84603 
                 0.83478 
                 0.82536 
                 0.81204 
                 0.80294 
                 0.79474 
               
               
                 709 nm 
                 0.88992 
                 0.87785 
                 0.86414 
                 0.85678 
                 0.84718 
                 0.83483 
                 0.82128 
                 0.81027 
                 0.79945 
               
               
                 710 nm 
                 0.89614 
                 0.88186 
                 0.87009 
                 0.86187 
                 0.85009 
                 0.83553 
                 0.82659 
                 0.82019 
                 0.80709 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3C 
               
             
            
               
                   
               
               
                 Normalized Excitation/Emission Spectrum of NIST SRM 2944 Glass 
               
               
                 (Excitation Range of 648 nm to 655 nm) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
                 Excitation λ 
               
               
                 Emission λ 
                 648 nm 
                 649 nm 
                 650 nm 
                 651 nm 
                 652 nm 
                 653 nm 
                 654 nm 
                 655 nm 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 660 nm 
                 0.22307 
                 0.21862 
                 0.21234 
                 0.20707 
                 0.20283 
                 0.19818 
                 0.19434 
                 0.19945 
               
               
                 661 nm 
                 0.23246 
                 0.22495 
                 0.22051 
                 0.21522 
                 0.21042 
                 0.20606 
                 0.20101 
                 0.19704 
               
               
                 662 nm 
                 0.24082 
                 0.23491 
                 0.22850 
                 0.22278 
                 0.21853 
                 0.21427 
                 0.20871 
                 0.20469 
               
               
                 663 nm 
                 0.25162 
                 0.24477 
                 0.23645 
                 0.23321 
                 0.22687 
                 0.22342 
                 0.21649 
                 0.21101 
               
               
                 664 nm 
                 0.26040 
                 0.25317 
                 0.24677 
                 0.24088 
                 0.23597 
                 0.23001 
                 0.22623 
                 0.21969 
               
               
                 665 nm 
                 0.26949 
                 0.26390 
                 0.25812 
                 0.25110 
                 0.24596 
                 0.23964 
                 0.23430 
                 0.22715 
               
               
                 666 nm 
                 0.28044 
                 0.27314 
                 0.26748 
                 0.25994 
                 0.25424 
                 0.24853 
                 0.24207 
                 0.23773 
               
               
                 667 nm 
                 0.29144 
                 0.28471 
                 0.27705 
                 0.27033 
                 0.26524 
                 0.25781 
                 0.25273 
                 0.24673 
               
               
                 668 nm 
                 0.30424 
                 0.29720 
                 0.28912 
                 0.28144 
                 0.27547 
                 0.26940 
                 0.26398 
                 0.25484 
               
               
                 669 nm 
                 0.31480 
                 0.30627 
                 0.29854 
                 0.29071 
                 0.28693 
                 0.28079 
                 0.27288 
                 0.26618 
               
               
                 670 nm 
                 0.32499 
                 0.31901 
                 0.31044 
                 0.30256 
                 0.29579 
                 0.29054 
                 0.28507 
                 0.27643 
               
               
                 671 nm 
                 0.33695 
                 0.33028 
                 0.32168 
                 0.31329 
                 0.30769 
                 0.29974 
                 0.29415 
                 0.28682 
               
               
                 672 nm 
                 0.35154 
                 0.34241 
                 0.33350 
                 0.32585 
                 0.31779 
                 0.31141 
                 0.30409 
                 0.29844 
               
               
                 673 nm 
                 0.36206 
                 0.35317 
                 0.34607 
                 0.33680 
                 0.32910 
                 0.32296 
                 0.31487 
                 0.30683 
               
               
                 674 nm 
                 0.37249 
                 0.36744 
                 0.35645 
                 0.34757 
                 0.34151 
                 0.33411 
                 0.32546 
                 0.31931 
               
               
                 675 nm 
                 0.38513 
                 0.37680 
                 0.37132 
                 0.36100 
                 0.35348 
                 0.34651 
                 0.33660 
                 0.33213 
               
               
                 676 nm 
                 0.39830 
                 0.38919 
                 0.38056 
                 0.37358 
                 0.36698 
                 0.35720 
                 0.34857 
                 0.33951 
               
               
                 677 nm 
                 0.40869 
                 0.40122 
                 0.39366 
                 0.38278 
                 0.37662 
                 0.36828 
                 0.36053 
                 0.35445 
               
               
                 678 nm 
                 0.42164 
                 0.41460 
                 0.40237 
                 0.39651 
                 0.38763 
                 0.37992 
                 0.37141 
                 0.36531 
               
               
                 679 nm 
                 0.43349 
                 0.42601 
                 0.41659 
                 0.40618 
                 0.39905 
                 0.39172 
                 0.38240 
                 0.37497 
               
               
                 680 nm 
                 0.45032 
                 0.44037 
                 0.43041 
                 0.42288 
                 0.41332 
                 0.40584 
                 0.39514 
                 0.38954 
               
               
                 681 nm 
                 0.46247 
                 0.45195 
                 0.44170 
                 0.43335 
                 0.42404 
                 0.41838 
                 0.40698 
                 0.39738 
               
               
                 682 nm 
                 0.47560 
                 0.46757 
                 0.45571 
                 0.44799 
                 0.43733 
                 0.42737 
                 0.42113 
                 0.41283 
               
               
                 683 nm 
                 0.48577 
                 0.48138 
                 0.46818 
                 0.46202 
                 0.45340 
                 0.44252 
                 0.43443 
                 0.42419 
               
               
                 684 nm 
                 0.50254 
                 0.49332 
                 0.48344 
                 0.47429 
                 0.46366 
                 0.45632 
                 0.44797 
                 0.43761 
               
               
                 685 nm 
                 0.51655 
                 0.50645 
                 0.49770 
                 0.48468 
                 0.47363 
                 0.46564 
                 0.45579 
                 0.45023 
               
               
                 686 nm 
                 0.52648 
                 0.51785 
                 0.50762 
                 0.49781 
                 0.48867 
                 0.48044 
                 0.46849 
                 0.45994 
               
               
                 687 nm 
                 0.54333 
                 0.53133 
                 0.52121 
                 0.50969 
                 0.50280 
                 0.49216 
                 0.48218 
                 0.47060 
               
               
                 688 nm 
                 0.55721 
                 0.54386 
                 0.53374 
                 0.52499 
                 0.51500 
                 0.50429 
                 0.49431 
                 0.48410 
               
               
                 689 nm 
                 0.56593 
                 0.55658 
                 0.54389 
                 0.53722 
                 0.52538 
                 0.51817 
                 0.50748 
                 0.49488 
               
               
                 690 nm 
                 0.58141 
                 0.57162 
                 0.55913 
                 0.55088 
                 0.53814 
                 0.53011 
                 0.51971 
                 0.50912 
               
               
                 691 nm 
                 0.59442 
                 0.58090 
                 0.57177 
                 0.56071 
                 0.55108 
                 0.54204 
                 0.53196 
                 0.52437 
               
               
                 692 nm 
                 0.60802 
                 0.59742 
                 0.58562 
                 0.57557 
                 0.56810 
                 0.55619 
                 0.54572 
                 0.53520 
               
               
                 693 nm 
                 0.61806 
                 0.61048 
                 0.60156 
                 0.58885 
                 0.57871 
                 0.56818 
                 0.56005 
                 0.55053 
               
               
                 694 nm 
                 0.63058 
                 0.62611 
                 0.60978 
                 0.60223 
                 0.59056 
                 0.58141 
                 0.57157 
                 0.56200 
               
               
                 695 nm 
                 0.64782 
                 0.63917 
                 0.62542 
                 0.61524 
                 0.60570 
                 0.59401 
                 0.58392 
                 0.57225 
               
               
                 696 nm 
                 0.66502 
                 0.65048 
                 0.64032 
                 0.62578 
                 0.61605 
                 0.60660 
                 0.59913 
                 0.58509 
               
               
                 697 nm 
                 0.67196 
                 0.66391 
                 0.65115 
                 0.64087 
                 0.63057 
                 0.61819 
                 0.60895 
                 0.59958 
               
               
                 698 nm 
                 0.68370 
                 0.67084 
                 0.66268 
                 0.65501 
                 0.64005 
                 0.63120 
                 0.62074 
                 0.60900 
               
               
                 699 nm 
                 0.69510 
                 0.68551 
                 0.67157 
                 0.66240 
                 0.65280 
                 0.64155 
                 0.62950 
                 0.62099 
               
               
                 700 nm 
                 0.70688 
                 0.69564 
                 0.68446 
                 0.67570 
                 0.66249 
                 0.65235 
                 0.63999 
                 0.62854 
               
               
                 701 nm 
                 0.71596 
                 0.70828 
                 0.69418 
                 0.68431 
                 0.67283 
                 0.66207 
                 0.64923 
                 0.64099 
               
               
                 702 nm 
                 0.72934 
                 0.71764 
                 0.70616 
                 0.69496 
                 0.68405 
                 0.67309 
                 0.66337 
                 0.65407 
               
               
                 703 nm 
                 0.73582 
                 0.72721 
                 0.71421 
                 0.70465 
                 0.69440 
                 0.68092 
                 0.67209 
                 0.66131 
               
               
                 704 nm 
                 0.74703 
                 0.73430 
                 0.72530 
                 0.71079 
                 0.70244 
                 0.69213 
                 0.68112 
                 0.67269 
               
               
                 705 nm 
                 0.75439 
                 0.74683 
                 0.73679 
                 0.72472 
                 0.71410 
                 0.70194 
                 0.69075 
                 0.67711 
               
               
                 706 nm 
                 0.76728 
                 0.75756 
                 0.74230 
                 0.72972 
                 0.72297 
                 0.71639 
                 0.69829 
                 0.69141 
               
               
                 707 nm 
                 0.77383 
                 0.76340 
                 0.75084 
                 0.74204 
                 0.73260 
                 0.71983 
                 0.71077 
                 0.69931 
               
               
                 708 nm 
                 0.78383 
                 0.77095 
                 0.76037 
                 0.75105 
                 0.73926 
                 0.72877 
                 0.71753 
                 0.70840 
               
               
                 709 nm 
                 0.79008 
                 0.78214 
                 0.76971 
                 0.75695 
                 0.74783 
                 0.73712 
                 0.72513 
                 0.71338 
               
               
                 710 nm 
                 0.79852 
                 0.78818 
                 0.77288 
                 0.76500 
                 0.75695 
                 0.74266 
                 0.73116 
                 0.72153 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Relative Absorption of Alexa Fluor ® 647 
               
               
                 vs. Excitation Wavelength 
               
            
           
           
               
               
               
            
               
                   
                 Excitation Wavelength 
                 AF 647 Relative 
               
               
                   
                 (nm) 
                 Absorption 
               
               
                   
                   
               
               
                   
                 630 
                 0.5403 
               
               
                   
                 631 
                 0.5666 
               
               
                   
                 632 
                 0.5917 
               
               
                   
                 633 
                 0.6202 
               
               
                   
                 634 
                 0.6501 
               
               
                   
                 635 
                 0.6780 
               
               
                   
                 636 
                 0.7052 
               
               
                   
                 637 
                 0.7364 
               
               
                   
                 638 
                 0.7706 
               
               
                   
                 639 
                 0.8041 
               
               
                   
                 640 
                 0.8279 
               
               
                   
                 641 
                 0.8537 
               
               
                   
                 642 
                 0.8796 
               
               
                   
                 643 
                 0.9080 
               
               
                   
                 644 
                 0.9325 
               
               
                   
                 645 
                 0.9504 
               
               
                   
                 646 
                 0.9666 
               
               
                   
                 647 
                 0.9826 
               
               
                   
                 648 
                 0.9920 
               
               
                   
                 649 
                 0.9977 
               
               
                   
                 650 
                 1.0000 
               
               
                   
                 651 
                 0.9956 
               
               
                   
                 652 
                 0.9905 
               
               
                   
                 653 
                 0.9747 
               
               
                   
                 654 
                 0.9590 
               
               
                   
                 655 
                 0.9391 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Relative Emission Intensity of Alexa Fluor ® 647 vs. Wavelength 
               
            
           
           
               
               
               
            
               
                   
                 Emission Wavelength 
                 Relative Emission 
               
               
                   
                 nm 
                 Intensity 
               
               
                   
                   
               
               
                   
                 660 
                 0.7259 
               
               
                   
                 661 
                 0.7660 
               
               
                   
                 662 
                 0.8055 
               
               
                   
                 663 
                 0.8477 
               
               
                   
                 664 
                 0.8856 
               
               
                   
                 665 
                 0.9075 
               
               
                   
                 666 
                 0.9320 
               
               
                   
                 667 
                 0.9507 
               
               
                   
                 668 
                 0.9738 
               
               
                   
                 669 
                 0.9841 
               
               
                   
                 670 
                 0.9973 
               
               
                   
                 671 
                 1.0000 
               
               
                   
                 672 
                 0.9928 
               
               
                   
                 673 
                 0.9827 
               
               
                   
                 674 
                 0.9688 
               
               
                   
                 675 
                 0.9522 
               
               
                   
                 676 
                 0.9390 
               
               
                   
                 677 
                 0.9057 
               
               
                   
                 678 
                 0.8844 
               
               
                   
                 679 
                 0.8615 
               
               
                   
                 680 
                 0.8339 
               
               
                   
                 681 
                 0.8042 
               
               
                   
                 682 
                 0.7709 
               
               
                   
                 683 
                 0.7475 
               
               
                   
                 684 
                 0.7092 
               
               
                   
                 685 
                 0.6830 
               
               
                   
                 686 
                 0.6567 
               
               
                   
                 687 
                 0.6199 
               
               
                   
                 688 
                 0.5930 
               
               
                   
                 689 
                 0.5717 
               
               
                   
                 690 
                 0.5372 
               
               
                   
                 691 
                 0.5095 
               
               
                   
                 692 
                 0.4887 
               
               
                   
                 693 
                 0.4652 
               
               
                   
                 694 
                 0.4451 
               
               
                   
                 695 
                 0.4267 
               
               
                   
                 696 
                 0.4064 
               
               
                   
                 697 
                 0.3899 
               
               
                   
                 698 
                 0.3731 
               
               
                   
                 699 
                 0.3564 
               
               
                   
                 700 
                 0.3406 
               
               
                   
                 701 
                 0.3303 
               
               
                   
                 702 
                 0.3180 
               
               
                   
                 703 
                 0.3064 
               
               
                   
                 704 
                 0.2973 
               
               
                   
                 705 
                 0.2845 
               
               
                   
                 706 
                 0.2749 
               
               
                   
                 707 
                 0.2693 
               
               
                   
                 708 
                 0.2625 
               
               
                   
                 709 
                 0.2528 
               
               
                   
                 710 
                 0.2494 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Relative Absorption of Alexa Fluor ® 635 
               
               
                 vs. Excitation Wavelength 
               
            
           
           
               
               
               
            
               
                   
                 Excitation Wavelength 
                 AF 635 Relative 
               
               
                   
                 (nm) 
                 Absorption 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 300 
                 0.446957202 
               
               
                   
                 301 
                 0.396653342 
               
               
                   
                 302 
                 0.343918215 
               
               
                   
                 303 
                 0.295014831 
               
               
                   
                 304 
                 0.248871208 
               
               
                   
                 305 
                 0.210934787 
               
               
                   
                 306 
                 0.179785525 
               
               
                   
                 307 
                 0.154627416 
               
               
                   
                 308 
                 0.133971426 
               
               
                   
                 309 
                 0.119256131 
               
               
                   
                 310 
                 0.105066014 
               
               
                   
                 311 
                 0.092018933 
               
               
                   
                 312 
                 0.08507731 
               
               
                   
                 313 
                 0.07738499 
               
               
                   
                 314 
                 0.070948979 
               
               
                   
                 315 
                 0.066119397 
               
               
                   
                 316 
                 0.061795427 
               
               
                   
                 317 
                 0.056687809 
               
               
                   
                 318 
                 0.052547137 
               
               
                   
                 319 
                 0.049170548 
               
               
                   
                 320 
                 0.045328507 
               
               
                   
                 321 
                 0.04315571 
               
               
                   
                 322 
                 0.040128211 
               
               
                   
                 323 
                 0.038543407 
               
               
                   
                 324 
                 0.038780253 
               
               
                   
                 325 
                 0.038624759 
               
               
                   
                 326 
                 0.036226444 
               
               
                   
                 327 
                 0.038645354 
               
               
                   
                 328 
                 0.037966741 
               
               
                   
                 329 
                 0.03680311 
               
               
                   
                 330 
                 0.037255176 
               
               
                   
                 331 
                 0.03745083 
               
               
                   
                 332 
                 0.036782515 
               
               
                   
                 333 
                 0.037184122 
               
               
                   
                 334 
                 0.037286068 
               
               
                   
                 335 
                 0.037224283 
               
               
                   
                 336 
                 0.037347854 
               
               
                   
                 337 
                 0.037430235 
               
               
                   
                 338 
                 0.037647515 
               
               
                   
                 339 
                 0.038295235 
               
               
                   
                 340 
                 0.038718467 
               
               
                   
                 341 
                 0.040220889 
               
               
                   
                 342 
                 0.041395847 
               
               
                   
                 343 
                 0.042836484 
               
               
                   
                 344 
                 0.043783865 
               
               
                   
                 345 
                 0.045607573 
               
               
                   
                 346 
                 0.04683196 
               
               
                   
                 347 
                 0.048283924 
               
               
                   
                 348 
                 0.049325013 
               
               
                   
                 349 
                 0.050344477 
               
               
                   
                 350 
                 0.051013822 
               
               
                   
                 351 
                 0.052280429 
               
               
                   
                 352 
                 0.053093941 
               
               
                   
                 353 
                 0.054082512 
               
               
                   
                 354 
                 0.055019595 
               
               
                   
                 355 
                 0.056027732 
               
               
                   
                 356 
                 0.057377749 
               
               
                   
                 357 
                 0.058983148 
               
               
                   
                 358 
                 0.060467034 
               
               
                   
                 359 
                 0.061568879 
               
               
                   
                 360 
                 0.063565587 
               
               
                   
                 361 
                 0.06554273 
               
               
                   
                 362 
                 0.067015289 
               
               
                   
                 363 
                 0.068705128 
               
               
                   
                 364 
                 0.070186955 
               
               
                   
                 365 
                 0.071350586 
               
               
                   
                 366 
                 0.072462729 
               
               
                   
                 367 
                 0.073389514 
               
               
                   
                 368 
                 0.073986776 
               
               
                   
                 369 
                 0.074636556 
               
               
                   
                 370 
                 0.07504846 
               
               
                   
                 371 
                 0.075469633 
               
               
                   
                 372 
                 0.07612868 
               
               
                   
                 373 
                 0.076315067 
               
               
                   
                 374 
                 0.075985544 
               
               
                   
                 375 
                 0.076880406 
               
               
                   
                 376 
                 0.076726972 
               
               
                   
                 377 
                 0.07677846 
               
               
                   
                 378 
                 0.077796894 
               
               
                   
                 379 
                 0.078806061 
               
               
                   
                 380 
                 0.079362132 
               
               
                   
                 381 
                 0.081236299 
               
               
                   
                 382 
                 0.082286655 
               
               
                   
                 383 
                 0.083790108 
               
               
                   
                 384 
                 0.085303857 
               
               
                   
                 385 
                 0.086354214 
               
               
                   
                 386 
                 0.086777446 
               
               
                   
                 387 
                 0.086087506 
               
               
                   
                 388 
                 0.085169988 
               
               
                   
                 389 
                 0.083501774 
               
               
                   
                 390 
                 0.081287787 
               
               
                   
                 391 
                 0.078620704 
               
               
                   
                 392 
                 0.075923758 
               
               
                   
                 393 
                 0.072740764 
               
               
                   
                 394 
                 0.069394039 
               
               
                   
                 395 
                 0.065687926 
               
               
                   
                 396 
                 0.062051838 
               
               
                   
                 397 
                 0.058406481 
               
               
                   
                 398 
                 0.055009297 
               
               
                   
                 399 
                 0.05188809 
               
               
                   
                 400 
                 0.048953269 
               
               
                   
                 401 
                 0.046184239 
               
               
                   
                 402 
                 0.044638567 
               
               
                   
                 403 
                 0.042949758 
               
               
                   
                 404 
                 0.042712913 
               
               
                   
                 405 
                 0.042549181 
               
               
                   
                 406 
                 0.043176305 
               
               
                   
                 407 
                 0.044144281 
               
               
                   
                 408 
                 0.045503567 
               
               
                   
                 409 
                 0.052867393 
               
               
                   
                 410 
                 0.048532096 
               
               
                   
                 411 
                 0.049653507 
               
               
                   
                 412 
                 0.051084875 
               
               
                   
                 413 
                 0.051868524 
               
               
                   
                 414 
                 0.052475054 
               
               
                   
                 415 
                 0.052331917 
               
               
                   
                 416 
                 0.051981798 
               
               
                   
                 417 
                 0.051003524 
               
               
                   
                 418 
                 0.049582453 
               
               
                   
                 419 
                 0.047851424 
               
               
                   
                 420 
                 0.04626662 
               
               
                   
                 421 
                 0.044124716 
               
               
                   
                 422 
                 0.041313467 
               
               
                   
                 423 
                 0.039376485 
               
               
                   
                 424 
                 0.036791783 
               
               
                   
                 425 
                 0.034980432 
               
               
                   
                 426 
                 0.032899284 
               
               
                   
                 427 
                 0.031241367 
               
               
                   
                 428 
                 0.029399123 
               
               
                   
                 429 
                 0.027843153 
               
               
                   
                 430 
                 0.027060535 
               
               
                   
                 431 
                 0.026195535 
               
               
                   
                 432 
                 0.024899065 
               
               
                   
                 433 
                 0.024362559 
               
               
                   
                 434 
                 0.023754999 
               
               
                   
                 435 
                 0.023806488 
               
               
                   
                 436 
                 0.022746863 
               
               
                   
                 437 
                 0.022489422 
               
               
                   
                 438 
                 0.021737696 
               
               
                   
                 439 
                 0.021623393 
               
               
                   
                 440 
                 0.02098494 
               
               
                   
                 441 
                 0.020500952 
               
               
                   
                 442 
                 0.02033722 
               
               
                   
                 443 
                 0.020048887 
               
               
                   
                 444 
                 0.019687441 
               
               
                   
                 445 
                 0.019410435 
               
               
                   
                 446 
                 0.018935715 
               
               
                   
                 447 
                 0.018514542 
               
               
                   
                 448 
                 0.018081012 
               
               
                   
                 449 
                 0.017495078 
               
               
                   
                 450 
                 0.01718512 
               
               
                   
                 451 
                 0.017031685 
               
               
                   
                 452 
                 0.016351013 
               
               
                   
                 453 
                 0.015918513 
               
               
                   
                 454 
                 0.015301686 
               
               
                   
                 455 
                 0.015095733 
               
               
                   
                 456 
                 0.014672501 
               
               
                   
                 457 
                 0.014488174 
               
               
                   
                 458 
                 0.014127757 
               
               
                   
                 459 
                 0.013766311 
               
               
                   
                 460 
                 0.013405894 
               
               
                   
                 461 
                 0.013417222 
               
               
                   
                 462 
                 0.012995019 
               
               
                   
                 463 
                 0.012892043 
               
               
                   
                 464 
                 0.012686091 
               
               
                   
                 465 
                 0.012531627 
               
               
                   
                 466 
                 0.012098097 
               
               
                   
                 467 
                 0.011984823 
               
               
                   
                 468 
                 0.011593514 
               
               
                   
                 469 
                 0.011378293 
               
               
                   
                 470 
                 0.01108996 
               
               
                   
                 471 
                 0.010760436 
               
               
                   
                 472 
                 0.010460776 
               
               
                   
                 473 
                 0.010513294 
               
               
                   
                 474 
                 0.010307341 
               
               
                   
                 475 
                 0.010163175 
               
               
                   
                 476 
                 0.010060198 
               
               
                   
                 477 
                 0.010019008 
               
               
                   
                 478 
                 0.009782163 
               
               
                   
                 479 
                 0.009524722 
               
               
                   
                 480 
                 0.009410419 
               
               
                   
                 481 
                 0.009380556 
               
               
                   
                 482 
                 0.00910252 
               
               
                   
                 483 
                 0.008936728 
               
               
                   
                 484 
                 0.008947026 
               
               
                   
                 485 
                 0.00884405 
               
               
                   
                 486 
                 0.008721508 
               
               
                   
                 487 
                 0.008752401 
               
               
                   
                 488 
                 0.008833752 
               
               
                   
                 489 
                 0.008927461 
               
               
                   
                 490 
                 0.009071627 
               
               
                   
                 491 
                 0.009349663 
               
               
                   
                 492 
                 0.009452639 
               
               
                   
                 493 
                 0.009668889 
               
               
                   
                 494 
                 0.009957222 
               
               
                   
                 495 
                 0.010213633 
               
               
                   
                 496 
                 0.010285716 
               
               
                   
                 497 
                 0.010523591 
               
               
                   
                 498 
                 0.010862383 
               
               
                   
                 499 
                 0.011192936 
               
               
                   
                 500 
                 0.01126502 
               
               
                   
                 501 
                 0.011512162 
               
               
                   
                 502 
                 0.011995121 
               
               
                   
                 503 
                 0.012314347 
               
               
                   
                 504 
                 0.012850853 
               
               
                   
                 505 
                 0.013324543 
               
               
                   
                 506 
                 0.013818829 
               
               
                   
                 507 
                 0.014385198 
               
               
                   
                 508 
                 0.014941269 
               
               
                   
                 509 
                 0.015310953 
               
               
                   
                 510 
                 0.016062679 
               
               
                   
                 511 
                 0.016670239 
               
               
                   
                 512 
                 0.01765881 
               
               
                   
                 513 
                 0.01830859 
               
               
                   
                 514 
                 0.019122102 
               
               
                   
                 515 
                 0.020306327 
               
               
                   
                 516 
                 0.021489524 
               
               
                   
                 517 
                 0.022808648 
               
               
                   
                 518 
                 0.024063928 
               
               
                   
                 519 
                 0.025599303 
               
               
                   
                 520 
                 0.027102755 
               
               
                   
                 521 
                 0.028935731 
               
               
                   
                 522 
                 0.030593647 
               
               
                   
                 523 
                 0.032591385 
               
               
                   
                 524 
                 0.034639581 
               
               
                   
                 525 
                 0.036410771 
               
               
                   
                 526 
                 0.038831741 
               
               
                   
                 527 
                 0.040675014 
               
               
                   
                 528 
                 0.042887972 
               
               
                   
                 529 
                 0.045257454 
               
               
                   
                 530 
                 0.047501305 
               
               
                   
                 531 
                 0.049591721 
               
               
                   
                 532 
                 0.05193031 
               
               
                   
                 533 
                 0.054257571 
               
               
                   
                 534 
                 0.056286202 
               
               
                   
                 535 
                 0.05850019 
               
               
                   
                 536 
                 0.060364058 
               
               
                   
                 537 
                 0.062247492 
               
               
                   
                 538 
                 0.06398779 
               
               
                   
                 539 
                 0.065666301 
               
               
                   
                 540 
                 0.066943206 
               
               
                   
                 541 
                 0.068456956 
               
               
                   
                 542 
                 0.069580426 
               
               
                   
                 543 
                 0.070702866 
               
               
                   
                 544 
                 0.072215586 
               
               
                   
                 545 
                 0.073399812 
               
               
                   
                 546 
                 0.074913562 
               
               
                   
                 547 
                 0.076458204 
               
               
                   
                 548 
                 0.078312805 
               
               
                   
                 549 
                 0.079991317 
               
               
                   
                 550 
                 0.082370066 
               
               
                   
                 551 
                 0.085067012 
               
               
                   
                 552 
                 0.087806178 
               
               
                   
                 553 
                 0.091400047 
               
               
                   
                 554 
                 0.095117486 
               
               
                   
                 555 
                 0.099360105 
               
               
                   
                 556 
                 0.104055818 
               
               
                   
                 557 
                 0.109421907 
               
               
                   
                 558 
                 0.115342007 
               
               
                   
                 559 
                 0.121850101 
               
               
                   
                 560 
                 0.128842183 
               
               
                   
                 561 
                 0.136432556 
               
               
                   
                 562 
                 0.144618132 
               
               
                   
                 563 
                 0.153289756 
               
               
                   
                 564 
                 0.162844915 
               
               
                   
                 565 
                 0.172514377 
               
               
                   
                 566 
                 0.18313225 
               
               
                   
                 567 
                 0.193594629 
               
               
                   
                 568 
                 0.204994091 
               
               
                   
                 569 
                 0.216249387 
               
               
                   
                 570 
                 0.228512819 
               
               
                   
                 571 
                 0.240241805 
               
               
                   
                 572 
                 0.25253716 
               
               
                   
                 573 
                 0.264133307 
               
               
                   
                 574 
                 0.275821102 
               
               
                   
                 575 
                 0.287219535 
               
               
                   
                 576 
                 0.298712705 
               
               
                   
                 577 
                 0.309184352 
               
               
                   
                 578 
                 0.319399588 
               
               
                   
                 579 
                 0.328667444 
               
               
                   
                 580 
                 0.3370703 
               
               
                   
                 581 
                 0.344207578 
               
               
                   
                 582 
                 0.350303767 
               
               
                   
                 583 
                 0.354978886 
               
               
                   
                 584 
                 0.358552159 
               
               
                   
                 585 
                 0.359890849 
               
               
                   
                 586 
                 0.360209045 
               
               
                   
                 587 
                 0.358386367 
               
               
                   
                 588 
                 0.355236326 
               
               
                   
                 589 
                 0.350581803 
               
               
                   
                 590 
                 0.345050953 
               
               
                   
                 591 
                 0.338203037 
               
               
                   
                 592 
                 0.330666211 
               
               
                   
                 593 
                 0.322880183 
               
               
                   
                 594 
                 0.315260976 
               
               
                   
                 595 
                 0.307969233 
               
               
                   
                 596 
                 0.301574413 
               
               
                   
                 597 
                 0.296158896 
               
               
                   
                 598 
                 0.292213879 
               
               
                   
                 599 
                 0.289752749 
               
               
                   
                 600 
                 0.289135921 
               
               
                   
                 601 
                 0.289979296 
               
               
                   
                 602 
                 0.292976933 
               
               
                   
                 603 
                 0.297548045 
               
               
                   
                 604 
                 0.304026276 
               
               
                   
                 605 
                 0.312582566 
               
               
                   
                 606 
                 0.323190141 
               
               
                   
                 607 
                 0.336041568 
               
               
                   
                 608 
                 0.350643589 
               
               
                   
                 609 
                 0.367674818 
               
               
                   
                 610 
                 0.386478267 
               
               
                   
                 611 
                 0.407578084 
               
               
                   
                 612 
                 0.430449092 
               
               
                   
                 613 
                 0.455286945 
               
               
                   
                 614 
                 0.48257666 
               
               
                   
                 615 
                 0.510771536 
               
               
                   
                 616 
                 0.541385322 
               
               
                   
                 617 
                 0.573123608 
               
               
                   
                 618 
                 0.607094417 
               
               
                   
                 619 
                 0.641138339 
               
               
                   
                 620 
                 0.677139839 
               
               
                   
                 621 
                 0.7121816 
               
               
                   
                 622 
                 0.7481831 
               
               
                   
                 623 
                 0.78335873 
               
               
                   
                 624 
                 0.818423146 
               
               
                   
                 625 
                 0.852023241 
               
               
                   
                 626 
                 0.883297105 
               
               
                   
                 627 
                 0.911862695 
               
               
                   
                 628 
                 0.937339 
               
               
                   
                 629 
                 0.959623043 
               
               
                   
                 630 
                 0.977274189 
               
               
                   
                 631 
                 0.989372859 
               
               
                   
                 632 
                 0.998094941 
               
               
                   
                 633 
                 1 
               
               
                   
                 634 
                 0.996438054 
               
               
                   
                 635 
                 0.98686127 
               
               
                   
                 636 
                 0.97186794 
               
               
                   
                 637 
                 0.951200622 
               
               
                   
                 638 
                 0.924982889 
               
               
                   
                 639 
                 0.893842894 
               
               
                   
                 640 
                 0.858419091 
               
               
                   
                 641 
                 0.820461045 
               
               
                   
                 642 
                 0.778570338 
               
               
                   
                 643 
                 0.734971257 
               
               
                   
                 644 
                 0.6888472 
               
               
                   
                 645 
                 0.643063994 
               
               
                   
                 646 
                 0.595664062 
               
               
                   
                 647 
                 0.549468951 
               
               
                   
                 648 
                 0.504469394 
               
               
                   
                 649 
                 0.461135991 
               
               
                   
                 650 
                 0.419945523 
               
               
                   
                 651 
                 0.379960905 
               
               
                   
                 652 
                 0.343331251 
               
               
                   
                 653 
                 0.308186513 
               
               
                   
                 654 
                 0.276006459 
               
               
                   
                 655 
                 0.245730435 
               
               
                   
                 656 
                 0.218813494 
               
               
                   
                 657 
                 0.193428838 
               
               
                   
                 658 
                 0.171083008 
               
               
                   
                 659 
                 0.150746244 
               
               
                   
                 660 
                 0.132704819 
               
               
                   
                 661 
                 0.116547858 
               
               
                   
                 662 
                 0.102007622 
               
               
                   
                 663 
                 0.089783321 
               
               
                   
                 664 
                 0.078539353 
               
               
                   
                 665 
                 0.068292194 
               
               
                   
                 666 
                 0.059540249 
               
               
                   
                 667 
                 0.052105369 
               
               
                   
                 668 
                 0.04514418 
               
               
                   
                 669 
                 0.039252913 
               
               
                   
                 670 
                 0.034093807 
               
               
                   
                 671 
                 0.029614344 
               
               
                   
                 672 
                 0.025949422 
               
               
                   
                 673 
                 0.02249869 
               
               
                   
                 674 
                 0.019667875 
               
               
                   
                 675 
                 0.017217042 
               
               
                   
                 676 
                 0.015260495 
               
               
                   
                 677 
                 0.01325246 
               
               
                   
                 678 
                 0.011459645 
               
               
                   
                 679 
                 0.01014155 
               
               
                   
                 680 
                 0.009071627 
               
               
                   
                 681 
                 0.007897699 
               
               
                   
                 682 
                 0.006847342 
               
               
                   
                 683 
                 0.005826848 
               
               
                   
                 684 
                 0.005076152 
               
               
                   
                 685 
                 0.004405777 
               
               
                   
                 686 
                 0.003849705 
               
               
                   
                 687 
                 0.00357167 
               
               
                   
                 688 
                 0.002840539 
               
               
                   
                 689 
                 0.002572801 
               
               
                   
                 690 
                 0.002161926 
               
               
                   
                 691 
                 0.001851968 
               
               
                   
                 692 
                 0.001502879 
               
               
                   
                 693 
                 0.001399902 
               
               
                   
                 694 
                 0.00119292 
               
               
                   
                 695 
                 0.000894289 
               
               
                   
                 696 
                 0.000606986 
               
               
                   
                 697 
                 0.00050298 
               
               
                   
                 698 
                 0.000420599 
               
               
                   
                 699 
                 0.000400004 
               
               
                   
                 700 
                 6.01824E−05 
               
               
                   
                 701 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Relative Emission of Alexa Fluor ® 635 
               
               
                 vs. Excitation Wavelength 
               
            
           
           
               
               
               
            
               
                   
                 Excitation Wavelength 
                 AF 635 Relative 
               
               
                   
                 (nm) 
                 Emission 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 604 
                 0 
               
               
                   
                 605 
                 0.027431 
               
               
                   
                 606 
                 0.017998 
               
               
                   
                 607 
                 0.019215 
               
               
                   
                 608 
                 0.02091 
               
               
                   
                 609 
                 0.023258 
               
               
                   
                 610 
                 0.027127 
               
               
                   
                 611 
                 0.032561 
               
               
                   
                 612 
                 0.038647 
               
               
                   
                 613 
                 0.043516 
               
               
                   
                 614 
                 0.050559 
               
               
                   
                 615 
                 0.058949 
               
               
                   
                 616 
                 0.067991 
               
               
                   
                 617 
                 0.080033 
               
               
                   
                 618 
                 0.09151 
               
               
                   
                 619 
                 0.1029 
               
               
                   
                 620 
                 0.123549 
               
               
                   
                 621 
                 0.139112 
               
               
                   
                 622 
                 0.158848 
               
               
                   
                 623 
                 0.179716 
               
               
                   
                 624 
                 0.210278 
               
               
                   
                 625 
                 0.2371 
               
               
                   
                 626 
                 0.267661 
               
               
                   
                 627 
                 0.303874 
               
               
                   
                 628 
                 0.342476 
               
               
                   
                 629 
                 0.377472 
               
               
                   
                 630 
                 0.424423 
               
               
                   
                 631 
                 0.465983 
               
               
                   
                 632 
                 0.504848 
               
               
                   
                 633 
                 0.556275 
               
               
                   
                 634 
                 0.597575 
               
               
                   
                 635 
                 0.653437 
               
               
                   
                 636 
                 0.6943 
               
               
                   
                 637 
                 0.741251 
               
               
                   
                 638 
                 0.785245 
               
               
                   
                 639 
                 0.836238 
               
               
                   
                 640 
                 0.865495 
               
               
                   
                 641 
                 0.901883 
               
               
                   
                 642 
                 0.932834 
               
               
                   
                 643 
                 0.954136 
               
               
                   
                 644 
                 0.979742 
               
               
                   
                 645 
                 0.989957 
               
               
                   
                 646 
                 0.999566 
               
               
                   
                 647 
                 0.999695 
               
               
                   
                 648 
                 1 
               
               
                   
                 649 
                 0.996131 
               
               
                   
                 650 
                 0.978916 
               
               
                   
                 651 
                 0.970612 
               
               
                   
                 652 
                 0.950181 
               
               
                   
                 653 
                 0.925314 
               
               
                   
                 654 
                 0.897187 
               
               
                   
                 655 
                 0.8698 
               
               
                   
                 656 
                 0.834891 
               
               
                   
                 657 
                 0.802113 
               
               
                   
                 658 
                 0.766769 
               
               
                   
                 659 
                 0.734991 
               
               
                   
                 660 
                 0.704212 
               
               
                   
                 661 
                 0.676042 
               
               
                   
                 662 
                 0.635264 
               
               
                   
                 663 
                 0.602313 
               
               
                   
                 664 
                 0.5681 
               
               
                   
                 665 
                 0.541495 
               
               
                   
                 666 
                 0.51002 
               
               
                   
                 667 
                 0.480893 
               
               
                   
                 668 
                 0.454419 
               
               
                   
                 669 
                 0.428379 
               
               
                   
                 670 
                 0.402556 
               
               
                   
                 671 
                 0.378646 
               
               
                   
                 672 
                 0.357867 
               
               
                   
                 673 
                 0.34013 
               
               
                   
                 674 
                 0.318828 
               
               
                   
                 675 
                 0.302047 
               
               
                   
                 676 
                 0.285789 
               
               
                   
                 677 
                 0.26753 
               
               
                   
                 678 
                 0.251533 
               
               
                   
                 679 
                 0.242272 
               
               
                   
                 680 
                 0.22684 
               
               
                   
                 681 
                 0.218666 
               
               
                   
                 682 
                 0.208494 
               
               
                   
                 683 
                 0.195713 
               
               
                   
                 684 
                 0.187975 
               
               
                   
                 685 
                 0.181454 
               
               
                   
                 686 
                 0.172065 
               
               
                   
                 687 
                 0.163109 
               
               
                   
                 688 
                 0.157284 
               
               
                   
                 689 
                 0.150806 
               
               
                   
                 690 
                 0.143981 
               
               
                   
                 691 
                 0.139417 
               
               
                   
                 692 
                 0.134678 
               
               
                   
                 693 
                 0.131461 
               
               
                   
                 694 
                 0.127027 
               
               
                   
                 695 
                 0.123636 
               
               
                   
                 696 
                 0.121288 
               
               
                   
                 697 
                 0.119202 
               
               
                   
                 698 
                 0.117637 
               
               
                   
                 699 
                 0.114681 
               
               
                   
                 700 
                 0.114072 
               
               
                   
                 701 
                 0.110768 
               
               
                   
                 702 
                 0.107812 
               
               
                   
                 703 
                 0.107551 
               
               
                   
                 704 
                 0.106508 
               
               
                   
                 705 
                 0.105378 
               
               
                   
                 706 
                 0.103986 
               
               
                   
                 707 
                 0.101465 
               
               
                   
                 708 
                 0.099683 
               
               
                   
                 709 
                 0.099074 
               
               
                   
                 710 
                 0.098596 
               
               
                   
                 711 
                 0.097987 
               
               
                   
                 712 
                 0.09577 
               
               
                   
                 713 
                 0.094422 
               
               
                   
                 714 
                 0.094118 
               
               
                   
                 715 
                 0.090597 
               
               
                   
                 716 
                 0.087076 
               
               
                   
                 717 
                 0.083772 
               
               
                   
                 718 
                 0.085076 
               
               
                   
                 719 
                 0.082381 
               
               
                   
                 720 
                 0.081294 
               
               
                   
                 721 
                 0.078207 
               
               
                   
                 722 
                 0.075294 
               
               
                   
                 723 
                 0.074208 
               
               
                   
                 724 
                 0.070556 
               
               
                   
                 725 
                 0.069165 
               
               
                   
                 726 
                 0.067513 
               
               
                   
                 727 
                 0.063253 
               
               
                   
                 728 
                 0.058731 
               
               
                   
                 729 
                 0.058949 
               
               
                   
                 730 
                 0.056384 
               
               
                   
                 731 
                 0.055297 
               
               
                   
                 732 
                 0.052341 
               
               
                   
                 733 
                 0.049385 
               
               
                   
                 734 
                 0.046211 
               
               
                   
                 735 
                 0.045081 
               
               
                   
                 736 
                 0.044864 
               
               
                   
                 737 
                 0.040082 
               
               
                   
                 738 
                 0.039864 
               
               
                   
                 739 
                 0.037778 
               
               
                   
                 740 
                 0.03643 
               
               
                   
                 741 
                 0.034778 
               
               
                   
                 742 
                 0.032952 
               
               
                   
                 743 
                 0.029996 
               
               
                   
                 744 
                 0.029474 
               
               
                   
                 745 
                 0.028605 
               
               
                   
                 746 
                 0.025736 
               
               
                   
                 747 
                 0.025301 
               
               
                   
                 748 
                 0.023649 
               
               
                   
                 749 
                 0.023867 
               
               
                   
                 750 
                 0.021475 
               
               
                   
                 751 
                 0.022388 
               
               
                   
                 752 
                 0.019389 
               
               
                   
                 753 
                 0.019389 
               
               
                   
                 754 
                 0.018215 
               
               
                   
                 755 
                 0.016302 
               
               
                   
                 756 
                 0.016694 
               
               
                   
                 757 
                 0.016346 
               
               
                   
                 758 
                 0.015694 
               
               
                   
                 759 
                 0.015215 
               
               
                   
                 760 
                 0.014737 
               
               
                   
                 761 
                 0.012911 
               
               
                   
                 762 
                 0.012129 
               
               
                   
                 763 
                 0.012042 
               
               
                   
                 764 
                 0.012737 
               
               
                   
                 765 
                 0.011172 
               
               
                   
                 766 
                 0.010999 
               
               
                   
                 767 
                 0.009868 
               
               
                   
                 768 
                 0.009912 
               
               
                   
                 769 
                 0.010607 
               
               
                   
                 770 
                 0.010477 
               
               
                   
                 771 
                 0.008695 
               
               
                   
                 772 
                 0.008868 
               
               
                   
                 773 
                 0.008868 
               
               
                   
                 774 
                 0.008129 
               
               
                   
                 775 
                 0.008955 
               
               
                   
                 776 
                 0