Patent Publication Number: US-7583381-B2

Title: Miniaturized fluorescence analysis system

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
   This application is a Divisional of U.S. application Ser. No. 11/217,567, filed Aug. 31, 2005 now abandoned, which claims the benefit of U.S. Provisional Application No. 60/606,000, filed Aug. 31, 2004. 

   GOVERNMENT LICENSE RIGHTS 
   This invention was made with government support under grant number 1-P50-HG002360-01 awarded by the National Institutes of Health. The government has certain rights in the invention. 

   BACKGROUND 
   1. Field 
   Embodiments of the present invention relate to fluorescence analysis systems and in particular to light emitting diode (LED)-based fluorescence analysis systems. 
   2. Discussion of Related Art 
   Fluorescence is the emission of light by molecules which have absorbed light. The fluorescing characteristics of such molecules (called fluorophors) are useful in detecting and tagging various microbiological events. The emission signal is shifted to higher wavelengths (Stokes-Shift) in relation to the excitation signal because the energy of the fluorescing light emitted is less than the light absorbed by the fluorophor. 
   Fluorometers exploit the fluorescing property of fluorophor molecules in the analysis of biological samples. The simulation and optimization of a LED-based fluorometer should ideally maximize the efficiency with which light is converted to signal (emission) and minimize bleed-through excitation light to the output signal path. In other words, the overlap between excitation light source and emission spectra should be minimized so that once the excitation light is optically filtered from the output signal, maximum emission signal remains for measurement. 
   Conventional fluorescence analysis systems use a laser or a high power white light source (e.g. Xenon lamp) to excite fluorophors in the sample under analysis. LEDs (light emitting diodes) are of interest to replace conventional light sources to increase the portability (reduce power, size, and weight) of the analysis system and to improve the flexibility of excitation spectra available to the user with reduced optics overhead. Several approaches have been used in LED-based fluorescence analysis systems. LEDs are an attractive alternative to conventional white light sources used in fluorescence analysis because of reduced power of operation, fewer imaging artifacts, and increased flexibility in spectral control without the need for high overhead optics. 
   General purpose, commercially available portable systems, such as the Turner Biosystems [1][2] instruments have used single LEDs to excite fluorescing samples; these systems rely on the user to select the LED to match the fluorophor or vice versa. Many results in the literature rely on single or small arrays of LEDs, where excitation bands are chosen close to the excitation spectra of the fluorophor and the resulting emission spectrum is optically filtered to minimize interference from the excitation signal. Still other approaches excite a sample using different LEDs at different times and subsequent signal processing to improve the extraction of the emission signal from the combined output signal. Finally, a variety of waveguides have been constructed to minimize the transfer of excitation light along the output emission path at the expense of reduced sample volume. 
   The use of LEDs, however, is often limited by three primary factors: (a) the broadband output of an LED often interferes with the measurement of emission signal; (b) the power (intensity) of light generated by an LED (mWatts) is often small compared to white light source (Watts) counterparts; and (c) the excitation peaks of the LED are often not well matched to the absorption efficiency of the fluorophor under analysis. The use of LEDs, for this reason, has been largely limited to high concentration applications where emitted fluorescence is sufficiently high (and noise sufficiently low) that LED limitations do not restrict effective measurements of the sample under analysis. The spectral flexibility, modularity, low-cost, and low power consumption of LEDs, however, continue to make them attractive options for fluorescence analysis, however. 
   In many approaches using LEDs, the choice of LED (or LEDs) is usually not optimized prior to the collection of data by the fluorescence analysis system. Instead, the optics and signal processing are assigned the task of separating excitation components from the emission signal in the output path. In addition, many LED-based fluorescence analysis systems used in commercial and research efforts are general-purpose. This means that they are suited to a relatively wide selection of fluorophors and the biological applications to which they are applied. 
   SUMMARY OF EMBODIMENTS OF THE INVENTION 
   Embodiments of the present invention are directed to automated and modular optimization of fluorescence analysis system that may maximize signal extraction (SNR) from an excited fluorophor. In one embodiment, the system includes an array of light emitting diodes (LEDs) that emit excitation light. The excitation light may have a first color and/or wavelength (blue, blue-green, green, purple, or other suitable color/wavelength. The color and/or wavelength of the excitation light of one LED may be different than the color and/or wavelength of another excitation light of one LED. 
   The system also includes control electronics that apply drive currents to the LEDs. The drive currents cause the LEDs to emit the excitation light. The drive current to one LED may be different than the drive current to another LED. For some embodiments, the drive current is greater than nominal drive current, greater than rated maximum current for the LED, and in may range between twenty and two hundred milliamps. The control electronics may include an emitter follower circuit and/or source follower circuit to drive the LEDs. 
   The control electronics also may pulse the drive current to the LEDs with signals having low duty cycles. For example, in one embodiment, the control electronics may pulse the drive current to the LEDs with signals having duty cycles between one percent and twelve percent. In other embodiments, the control electronics may pulse the drive current to the LEDs with signals having duty cycles at or greater than twelve percent. 
   The system also includes optics to couple excitation light from the LEDs to a holder for the fluorophor. In one embodiment, an optical fiber bundle may be coupled to each individual LED so that each LED has its own optical fiber bundle associated with it. The optical fiber bundles may be bundled together so that the excitation light from the LEDs may be aggregated into a single light beam that has a substantially uniform intensity profile and/or a substantially uniform wavelength distribution. The bundle of bundles couples the single light beam to the fluorophor holder. In one embodiment, the optical fiber bundles are bundled together in a random manner. 
   For some embodiments, the system may include a beam splitter to split off a small portion of the single light beam and to direct the small portion to circuitry to measure the intensity of the small portion of the single light beam as a function of the color and/or wavelength. The circuitry may adjust drive current to one or more LEDs in response to the measured intensity of the small portion of the single light beam. The circuitry may be a spectrophotometer. 
   For other embodiments, one or more PIN diode may be coupled to one or more LEDs, respectively, to detect the excitation light emitted from the LED. There may be circuitry to adjust one or more drive currents in response to the detected excitation light. 
   When the excitation light impinges on a fluorophor placed in the fluorophor holder, the fluorophor may emit light that in response to the excitation light. The emitted light may have a color and/or wavelength that is different than the color and/or wavelength of the excitation light. In one embodiment, the fluorophor holder may be a cuvette. 
   The system also includes a photodetector to detect light emitted from the fluorophor. The photodetector may a photomultiplier tube, an avalanche photodiode, photodiode, phototransistor, and/or a charge-coupled device (CCD). 
   The system also includes optical fiber to couple the light emitted from the fluorophor to the photodetector. 
   For some embodiments, the system may be used to select a configuration for the LED array. The system may determine at least two possible permutations of LEDs for the LED array and for each permutation determine a total excitation light that is to be emitted from the LED array, determine an amount of excitation light that is to reach the fluorophor based on the total excitation light emitted from the LED array, determine an amount of light that is to be transmitted through the fluorophor based on the amount of excitation light that is to reach the fluorophor, based on an amount of attenuation in an emission path to a photodetector from the fluorophor in the fluorescence analysis system, and based on filtering of the light that is to be transmitted through the fluorophor, determine an amount of light that is to be emitted by the fluorophor based on the amount of excitation light that is to reach the fluorophor and based on the amount of light that is to be transmitted through the fluorophor, determine an amount of light that is to reach the photodetector based on the amount of light that is to be emitted by the fluorophor, and determine a leakage penalty for the fluorescence analysis system based on the amount of light that is to be transmitted through the fluorophor and based on the amount of light that is to reach the photodetector. The system then compares the leakage penalties for each permutation of LEDs in the LED array and ranks the permutations of LEDs in the LED array based on the comparison of their respective leakage penalties. 
   In one embodiment, the system may receive from a user information associated with a type of LEDs to be selected from a database, information associated with a number of LEDs to be placed in the LED array, information associated with the fluorophor of interest, information associated with at least one undesirable fluorophor, information associated with a minimum concentration detection capability for the fluorophor of interest, and/or information associated with a time frame within which to perform fluorescence analysis on the fluorophor of interest. 
   The system may, for each permutation of LEDs in the LED array, determine optical power and/or spectral shape of an aggregate output of the LED array, receive information associated with spectral characteristics of the photodetector, calculate output current and/or output voltage of the photodetector based on the information associated with the spectral characteristics of the photodetector, determine a drive current for each LED in the LED array based on the leakage penalty, determine a duty cycle for the drive current based on the leakage penalty, and/or determine a duty cycle in the range of one percent to twelve percent for the drive current based on the leakage penalty. 
   For some embodiments, optimizing software may be used to perform the above-described method. In these embodiments, the software may be a machine-readable medium having data to cause a machine to select a configuration for the LED array. 
   As will be described below, the optimization software takes into account a variety of factors of practical fluorescence analysis including optics, photodetector properties, attenuation in the sample itself, and leakage of the excitation signal to the output signal path. While most fluorometer systems use a single light source such as a Xenon or Mercury lamp, in embodiments of the present invention the optimization software relies on a combination of several LEDs in an array driven at various nominal and overdrive currents. Overdriving the LEDs enables spectral shifts from nominal peak excitation wavelengths that increase the flexibility and intensity of spectra identified by optimization software. Given a fluorophor, environmental conditions, and optical path constraints, the optimization software may generate the best possible combination of LEDs to excite the sample and maximize the emission signal collected in the output path. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally equivalent elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the reference number, in which: 
       FIG. 1  is a high-level block diagram illustrating a miniaturized fluorescence analysis system according to an embodiment of the present invention; 
       FIG. 2  is a schematic diagram illustrating the LED driver depicted in  FIG. 1  according to an embodiment of the present invention; 
       FIG. 3  is graphical representation illustrating experimental measurements of various commercially available LEDs across time and drive current to characterize their effective lifetime according to an embodiment of the present invention; 
       FIG. 4  is graphical representation illustrating experimental demonstration of increased lifetime of the reduced duty cycle according to an embodiment of the present invention; 
       FIG. 5  is a graphical representation illustrating an example of the effectiveness of LED as compared to Xenon excitation according to an embodiment of the present invention; 
       FIGS. 6A and 6B  illustrate a flowchart of operation of the system depicted in  FIG. 1  according to an embodiment of the present invention; 
       FIG. 7 , including  7 ( a ),  7 ( b ), and  7 ( c ) is a graphical representation illustrating results generated by the process depicted in  FIG. 6  for evaluating GFPuv according to an embodiment of the present invention; 
       FIG. 8  is a graphical representation of an emission spectrum according to an embodiment of the present invention; 
       FIG. 9  is a graphical representation of the results of LED array optimization according to an embodiment of the present invention; 
       FIG. 10A and 10B  are graphical representations of the results of Rhodamine analysis according to an embodiment of the present invention; and 
       FIG. 11  is a graphical representation of an intensity comparison according to an embodiment of the present invention. 
   

   DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS 
     FIG. 1  is a high-level block diagram illustrating a miniaturized fluorescence analysis system  100  according to an embodiment of the present invention, in which an LED array  102  is coupled to fiber optic coupling  104 . The fiber optic coupling  104  is optionally coupled to a beam splitter  106 , which is part of a feed back loop. The example fiber optic coupling  104  may be coupled to a sample  108  (e.g., fluorophor) without the feed back loop. The sample  108  is coupled to fiber optic coupling  110 , which is optionally coupled to one or more optical dispersion devices  112 . The fiber optic coupling  110  may be coupled to a photodiode array  114  without the optical dispersion devices  112 . The photodetector  114  (shown as a photodiode array) is coupled to a signal processing module  116 . A monitor light source module  118  is coupled in the feed back loop between the beam splitter  106  and the LED array  102 . The illustrated module  118  includes an LED driver  120 . 
   LEDs are traditionally less stable than their white light source counterparts. In the system  100 , the real-time feedback loop monitors the LED array  102  output and adjusts the LED array  102  drive currents and output powers to maintain a stable signal into the fluorophor sample  108 . The output light passes from the LED array  102  through the randomizing fiber optic coupling  104 , and into the beam splitter  106 , where most of the light travels to the sample  108  (for exciting the fluorophor or other luminescing sample). A small percentage of the light may be fed back to a spectrophotometer in the monitor light source module  118  where it is analyzed for power and spectral properties, for example. The spectrophotometer sends power and spectral information to a microcontroller in the monitor light source module  118 , which is then used to adjust the LED array  102  output properties through the LED driver  120 . 
   For some embodiments, the LED array  102  is a highly flexible, modular LED array having several LEDs of different types (e.g., purple, blue, blue-green, and green) operating under several different possible drive currents (e.g., 5 and 20 mA are nominal operating conditions and 50, 100, and 150 mA are overdrive currents that not only increase the intensity of LED output but also produce fine shifts (in wavelength) of the spectral properties of the LED). In one embodiment, using type and drive current as variables, the 5-element LED array can produce a maximum of 3.2 million possible aggregate spectra. 
   The LED driver  120  is designed to be a dynamic, feedback driven light source suitable for use in fluorescence analysis systems. It is designed as a solution to common drawbacks of conventional fluorescence light sources (large cost, power, and size) and resolves issues associated with using LEDs as light sources for fluorescence analysis. (low intensity, stability, and lifetime, poor spectral control). An obstacle to using LEDs as a light source for fluorescence analysis is the small light intensity produced as compared to a conventional light source (Xenon lamp). The LED driver  120  solves this problem by overdriving the LEDs in the LED array  102  with very high currents, up to and beyond ten times the rated maximum current for the LED (20 mA DC), for example. Normal operation at these currents using DC may destroy the LED, but the LED driver  120  has solved this problem by pulsing the LEDs in the LED array  102  at these high currents for very short periods of time, in the range of 1-12% duty cycle, for example. Light intensity out of an LED is proportional to the input current, therefore each pulse of the LED may produce ten times the normal light intensity produced under manufacturer&#39;s operation specifications. By pulsing the LEDs in this way, lifetime degradation due to high currents may be reduced. 
   An additional side-effect of pulsing an LED at a high current with a low duty cycle is a shift in the spectral output of the LED. As mentioned above, one of the key problems encountered when using LEDs as a light source for fluorescence analysis is poor spectral control of the LED light output. Limited output spectra are available because LEDs are available only in discrete fixed wavelengths, as well as a few broadband LEDs. Creating a broadband light source from LEDs is difficult because of the extreme difference between types of LEDs. Some colors have higher intensities than others, “white light” LEDs are predominantly blue, and there can be both spectral and intensity differences between two LEDs of the same color and type. 
   These inconsistencies are solved with the LED driver  120 , which adjusts intensity variations using driving current. In this manner, the spectral shift side-effect mentioned above, which is usually regarded as an inhibitor to using LEDs as light sources, is exploited to adjust the spectral output of the LEDs to match the light source needs of the application. By driving an array of LEDs, aggregated into a single light source, the LED driver  120  creates a tunable light source. 
   In one embodiment, the LED driver  120  may be a printed circuit board (PCB) having four high current driver circuits as shown in  FIG. 2 , which is a schematic diagram of the LED driver  120  according to an embodiment of the present invention. The illustrated LED driver  120  may be capable of controlling a pair of LEDs at user adjustable duty cycles between one percent and twelve percent and at current levels between ten milliamps and two hundred milliamps. In the LED driver  120 , an emitter-follower (or source follower if using CMOS technology) supplies high current to the driving transistor Q 1 . A crystal oscillator (not shown) and a series of counters (not shown) may supply the low duty cycle driving signal to the LED drivers  120 . 
   Various commercially available LEDs (blue, blue-green, purple, green) have been experimentally measured across time and drive current to characterize their effective lifetime. An example of the characterization results is shown in  FIG. 3 . The higher the drive current, the shorter the lifetime of the LED. However, lifetime degradation can be circumvented, for fluorescence analysis applications, by reducing the duty cycle of the LED, so that they are activated long enough to excite fluorescence, but not so long as to prematurely degrade the useful lifetime of the LED. 
   Experimental demonstration of increased lifetime of the reduced duty cycle approach is shown in  FIG. 4  for DC operation (100% duty cycle) and low (1%) duty cycle operation. Lifetime of the blue LED shown in these results improves by an average of 30% using the LED driver  120 . 
   Using high drive currents, it is possible to achieve the power levels of conventional light sources in practical biological analysis experiments. An example of the effectiveness of LED as compared to Xenon excitation is shown in  FIG. 5 . This experiment captures the fluorescence of AM1 bacteria tagged with UV-GFP fluorophor. An array of 7 purple LEDs operating at 50 mA currents are used to excite the UV-GFP tagged bacteria at comparable levels to the Xenon light source in a standard Shimadzu RF3401 fluorometer. Standard growth studies are typically performed over a 24 hour period with data collection occurring every 3 hours. These experiment lengths cannot be accommodated with LEDs at high currents, using DC operation, but fall well within the limits of LEDs operating at the reduced duty cycles controlled by the LED driver module described herein. These types of electronic interface and control modules are essential to the effective use of LEDs and LED arrays in low-level fluorescence analysis. 
   The fiber optic coupling  104  includes an optical fiber bundle coupled to each individual LED so that each LED. The optical fiber bundles are bundled together in a random manner to aggregate the excitation light into a single light beam. Without the random fiber optic coupling  104  the output of the LED array  102  might be a series of discrete spots that may excite the sample  108  in an unpredictable and unstable manner. 
   The sample  108  may be held in a standard, commercially available cuvette holder, but may be coupled within the system  100  via the fiber optic coupling  104  and the fiber optic coupling  110  rather than traditional optical components. The fiber optic couplings  104  and  110  may be any suitable single mode or multimode optical fibers. The fiber optic couplings  104  and  110  also reduce or eliminate the need for periodic or unpredictable realignment of optics. 
   The photodetector  114 , although shown as photodiodes, may be any conventional photomultiplier tubes, avalanche photodiodes, low power photodiodes, phototransistors, charge-coupled devices that are compatible with portable, small footprint systems. Alternative photodetection schemes may be enabled by the reduced optical losses in the system  100  incurred by the use of fiber optic couplings  104  and  110  rather than discrete optics an the absence of slits required for white light sources, which also decrease optical throughput. 
   The LED configuration may be optimized using a design simulation program that accounts for LED output characteristics, optical filtering and losses, photodetector properties, sample characteristics, sample attenuation and other factors in seeking the “best” LED configuration in terms of overlap between the emission and excitation spectra, total emission intensity (SNR), or both. 
     FIG. 6  is a flowchart of a process  600  illustrating operation of the system  100  according to an embodiment of the present invention. The process  600  may be implemented using hardware, software, or a combination thereof. In implementations using software, the software may be stored on a machine-accessible medium. In a block  602  the process  600  begins and control passes to a block  604 . 
   In a block  604 , the process  600  determines at least two possible permutations of LEDs for the LED array  102 . 
   In a block  606 , for each permutation of LEDs in the LED array, the process  600  determines a total excitation light I IN  (λi) that is to be emitted from the permutation of LEDs to be placed in the LED array  102 . 
   In a block  608 , the process  600  determines an amount of excitation light that is to reach the fluorophor sample  108  based on the total excitation light emitted from the permutation of LEDs. At a particular wavelength, λi, the light that reaches the sample  108  may be expressed as I S (λi):
 
 I   S (λ i )= I   IN (λ i )× A   L   ×F   L (λ i )  (1)
 
   where I IN (λi) is the total excitation light (Watts) impinging on the sample at wavelength λi, that is attenuated by the optical loss in the excitation path A L  and by F L (λi), the transmissivity of filter in the excitation path. 
   In a block  610 , the process  600  determines an amount of light that is to be transmitted through the fluorophor sample  108  based on the amount of excitation light that is to reach the fluorophor sample  108 , the amount of attenuation in the emission path to the photodetector array  114  from the fluorophor sample  108 , and based on filtering of the light that is to be transmitted through the fluorophor sample  108 . The light that is transmitted through the sample, rather than absorbed by the fluorophor can be expressed, according to Beer&#39;s Law, in terms of the extinction coefficient ε(λi) to obtain I L     —     max (λi):
 
 I   L     —     max (λ i )= I   S (λ i )×(10 −ε(λ     i     )cL )  (2)
 
   where ε(λi) is the extinction coefficient (liters/cm*mole) at wavelength λi and is given by the excitation spectrum for the fluorophor sample  108 , c is the concentration of fluorophor sample  108  in the sample (moles per liter); and L is the fluorophor sample  108  path length (cm). 
   The transmitted light may then be attenuated by optical losses (A E ) and filtered (F E (λi)) in the emission path to obtain I L (λi):
 
 I   L (λ i )= I   L     —     max (λ i )× A   S   ×A   E   ×F   E (λ i )  (3)
 
   The factor A S  accounts for scattering and other losses between the sample and the output optical path. The light that is not transmitted through the fluorophor sample  108  is absorbed. 
   In a block  612 , the process  600  determines an amount of light that is to be emitted by the fluorophor sample  108  based on the amount of excitation light that is to reach the fluorophor sample  108  and the amount of light that is to be transmitted through the fluorophor sample  108 . Some of this absorbed light is then emitted as fluorescence I E :
 
 I   E =( I   S   −I   L     —     max )× Q   (4)
 
   where the factor Q represent fluorescence efficiency (quantum yield), and is a function of such variables as pH, temperature, and heavy metal ion concentration; I S  and I L max represent the total light reaching the sample  108  and transmitted light, respectively, integrated across all wavelengths λi. The total emitted light I E  is redistributed across wavelength λi according to the emission spectrum of the fluorophor sample  108  (available from the manufacturer). 
   In a block  614 , the process  600  determines an amount of light that is to reach the photodetector array  114  based on the amount of light that is to be emitted by the fluorophor sample  108 . The fluorescence signal that reaches the photodetector array  114  at the end of the optical path is then given by I E ′:
 
 I   E ′(λ i )= I   E (λ i )× A   E   ×F   E (λ i )  (5)
 
   In a block  616 , the process  600  determines a leakage penalty for the fluorescence analysis system  100  based on the amount of light that is to be transmitted through the fluorophor sample  108  and based on the amount of light that is to reach the photodetector array  114 . In order to optimize fluorescence emission, a performance metric P is defined as follows: 
   
     
       
         
           
             
               
                 
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   This dimensionless parameter is a measure of how much leakage from the excitation process affects the emission signal and is analogous to the reciprocal of SNR (signal to noise ratio) in electronic circuits. This penalty P can then be integrated across the visible light spectrum: 
   
     
       
         
           
             
               
                 P 
                 = 
                 
                   
                     ∫ 
                     
                       λ 
                       min 
                     
                     
                       λ 
                       max 
                     
                   
                   ⁢ 
                   
                     
                       P 
                       ⁡ 
                       
                         ( 
                         
                           λ 
                           i 
                         
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   It is this metric that is optimized (reduced and/or minimized) by the process  600 . Ultimately, this metric includes effects of significant interferents as well as bleed-through from the excitation signal. These factors that attenuate the signal in the excitation path, sample, emission path, and filtering properties of the excitation and emission paths can be adjusted by the user. The process  600  uses these parameters in combination with the targeted fluorophor and sample  108  characteristics to generate a ranked series of LED configurations that reduce the penalty metric P. 
   In a block  618 , the process  600  compares the leakage penalties for each permutation of LEDs in the LED array  102 . Results generated by the process  600  for evaluating GFPuv are shown in  FIG. 7  for (a) an optimized excitation spectrum, and (b) a non-optimized configuration. 
   In a block  620 , the process  600  ranks the permutations of LEDs in the LED array  102  based on the comparison of their respective leakage penalties. 
   In a block  622 , for each permutation of the LEDs in the LED array  102  the process  600  determines a duty cycle in the range of one percent to twelve percent for the drive current based on the leakage penalty. 
   In a block  624 , for each permutation of the LEDs in the LED array  102  the process  600  determines a drive current for each LED in the LED array  102  based on the leakage penalty. In one embodiment, the drive current is significantly in excess of a rated maximum current for the at least one LED in the LED array  102 . In a process  626 , the process  600  finishes. 
   In one embodiment, the process  600  has been optimized in a representative application for the excitation of GFPuv and has been compared to a single LED light source. The simulation parameters are chosen for proof-of-concept only. Although most fluorescence systems use some sort of optical filtering to reduce the interference of excitation with emission signal, it is possible, within the flexibility and modularity of the process  600  that some applications (that measure spectra rather than total intensity) will not require optical filtering and hence will not suffer from the optical losses associated with such filtering. Interfering effects such as autofluorescence and Raman scattering can be implemented into the current the process  600  and may be used to optimize even more accurately the system design to the needs of the targeted application. 
   Currently, the system  100  has a database consisting of several LEDS (blue, blue-green, green and purple) operating at drive currents from 1 mA to 100 mA (corresponding to spectral shifts from 0 nm to 10 nm from the peak wavelength at nominal operation). The penalty metric described in the previous section is minimized to determine the optimal combination of LEDs for each application. 
   The process  600  rates a range of optimizing LED combinations by comparing penalty calculations. The penalty for the best configuration is calculated at 0.159, compared to the less efficient non-optimized configuration at 0.365. The emission spectra resulting from the excitation by the closest matching single LED is compared to the GFPuv absorption spectra in  FIG. 7(   c ). From the data plotted in these figures, it can be shown that the optimized spectrum from the LED array  102  reduces the excitation signal leakage into the output path and improves the emission signal that can be collected for interpretation. Performance improvements over single LED excitation show a 70.1% increase in collected emission signal for GFPuv fluorophors. The best non-optimized and single LED configurations are shown in Table 1 below. 
   
     
       
         
             
           
             
               TABLE 1 
             
           
          
             
                 
             
             
               Configurations for GFPuv fluorophors 
             
          
         
         
             
             
             
             
          
             
                 
               Configuration 
               LEDs 
               Current 
             
             
                 
                 
             
             
                 
               Best 
               1 purple 
               10 mA 
             
             
                 
                 
               2 purple 
               20 mA 
             
             
                 
                 
               2 purple 
               50 mA 
             
             
                 
               Non-optimized 
               1 blue 
               10 mA 
             
             
                 
                 
               1 blue 
               100 mA  
             
             
                 
                 
               2 purple 
               20 mA 
             
             
                 
                 
               1 purple 
               10 mA 
             
             
                 
               Single LED 
               1 purple 
               50 mA 
             
             
                 
                 
             
          
         
       
     
   
     FIG. 8  compares all three emission spectrum from  FIG. 7  to demonstrate the analysis that is achieved by the process  600  in determining the optimal configuration of LEDs in the LED array  102 . A 70.4% improvement in the excitation signal over the single LED is achieved by the best configuration. These improvements support the use of design optimization of process  600  prior to the experimentation and fluorescent signal analysis. 
   In an alternative embodiment, the optimal configuration provided by the process  600  may be implemented in a 5-element LED array  102  with the LED driver  120  to reduce the duty cycle to the LEDs (for improving reliability and lifetime of the over driven LEDs). The outputs of the LEDs may then be coupled into a randomized fiber optic bundle to provide a uniform input to the sample under analysis (eliminating the imaging artifacts common to many white light sources). In this experimental embodiment, the LED array  102  and fiber optic bundle  104  may be coupled into a Shimadzu RF3401 fluorometer and the outputs measured in a manner comparable to when a Xenon source is used for sample  108  excitation. In one embodiment, the LED configuration is optimized for three representative applications as follows: (1) a theoretical desired spectral output; (2) the excitation of Rhodamine dye; and (3) the excitation of bacteria; GFPuv is cloned into Methylobacterium extorquens AM123, such that when suspended in liquid media, the fluorescent intensity of the cells is indicative of the cell concentration. 
   To demonstrate the capability of this LED array  102  over its closest alternatives, a desired spectrum is arbitrarily generated as shown in  FIG. 9 . Three possibilities for matching the LED spectrum to actual LED output are shown: (a) the closest single LED; (b) the closest combination of four types of LEDs operating under nominal conditions (with no spectral shift); and (c) the closest combination of four types of LEDs operating under nominal and overdrive conditions as described in the previous section. Qualitatively, this figure shows that the best match between desired and actual spectrum is achieved by the more flexible array (the one consisting of LEDs operating at nominal and overdrive conditions). The best matches (as measured by the error between desired and actual spectra) are configured as follows for each type of LED array as shown in Table 2. 
   The best, non-optimized, and single LED configurations are shown in Table 2 below. 
   
     
       
         
             
           
             
               TABLE 2 
             
           
          
             
                 
             
             
               Configurations for GFPuv fluorophors 
             
          
         
         
             
             
             
             
          
             
                 
               Configuration 
               LEDs 
               Current 
             
             
                 
                 
             
             
                 
               5 LEDs at nominal operation 
               1 blue 
               10 mA 
             
             
                 
                 
               1 blue 
               10 mA 
             
             
                 
                 
               1 blue 
               20 mA 
             
             
                 
                 
               1 blue-green 
               10 mA 
             
             
                 
                 
               1 blue-green 
               20 mA 
             
             
                 
               5 LEDs in overdrive 
               1 blue 
               10 mA 
             
             
                 
                 
               1 blue 
               20 mA 
             
             
                 
                 
               1 blue 
               20 mA 
             
             
                 
                 
               1 blue-green 
               20 mA 
             
             
                 
                 
               1 blue-green 
               100 mA  
             
             
                 
               Single LED 
               1 blue 
               50 mA 
             
             
                 
                 
             
          
         
       
     
   
   To complement these simulation results, the optimization process  600  and LED array  102  have been tested on two representative applications. The first application demonstrates that the LED array  102  is capable of producing an emission signal within 25% of the performance of the Xenon source for a biological analysis application ( FIG. 11 ). The bacteria Methylobacterium extorquens AM123 is analyzed under identical experimental conditions for both the Xenon source and a five element LED array  102  consisting of purple LEDs operating at 20 mA (nominal operation) and purple LEDs operating at 50 mA (overdrive operation). While nominal operation does not enable the LEDs to approach the power output of the Xenon lamp, overdrive operation brings the LED generated emission output within 25% of that produced by the Xenon source. 
   To demonstrate the increased efficiency of the LED array  102  presented here, the emission signal for the (a) single LED array and (b) optimized LED array is presented for the excitation of Rhodamine dye ( FIG. 10 ). Despite the fact that the single LED array  102  (the closest match of a single LED excitation peak to the Rhodamine dye absorption characteristics) has a much greater intensity than the optimized LED array  102 , the emission signal improves twofold over the non-optimized, single LED array  102 . The optimized LED array  102  consists of two blue LEDs operating at 5 mA and three purple LEDs operating at 5 mA. 
   Embodiments of the present invention may be implemented using hardware, software, or a combination thereof. In implementations using software, the software may be stored on a machine-accessible medium. A machine-accessible medium includes any mechanism that may be adapted to store and/or transmit information in a form accessible by a machine (e.g., a computer, network device, personal digital assistant, manufacturing tool, any device with a set of one or more processors, etc.). For example, a machine-accessible medium includes recordable and non-recordable media (e.g., read only memory (ROM), random access memory (RAM), magnetic disk storage media, optical storage media, flash memory devices, etc.), as recess as electrical, optical, acoustic, or other form of propagated signals (e.g., carrier waves, infrared signals, digital signals, etc.). 
   In the above description, numerous specific details, such as, for example, particular processes, materials, devices, and so forth, are presented to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the embodiments of the present invention may be practiced without one or more of the specific details, or with other methods, components, etc. In other instances, structures or operations are not shown or described in detail to avoid obscuring the understanding of this description. 
   Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, process, block, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification does not necessarily mean that the phrases all refer to the same embodiment. The particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. 
   The terms used in the following claims should not be construed to limit embodiments of the invention to the specific embodiments disclosed in the specification and the claims. Rather, the scope of embodiments of the invention is to be determined entirely by the following claims, which are to be construed in accordance with established doctrines of claim interpretation.