Patent Publication Number: US-2003230728-A1

Title: Multiwavelength transilluminator for absorbance and fluorescence detection using light emitting diodes

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
     [0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/405,843, filed Aug. 26, 2002, entitled MULTIWAVELENGTH TRANSILLUMINATOR FOR ABSORBANCE AND FLUORESCENCE DETECTION USING LIGHT EMITTING DIODES, and U.S. Provisional Patent Application Serial No. 60/388,191, filed Jun. 13, 2002, entitled AUTOMATED PROTEIN GEL PROCESSING METHODS AND SYSTEM, the disclosures of which are incorporated herein by reference in their entirety. 
    
    
     
       FIELD OF THE INVENTION  
       [0002] The present invention is directed to a method and apparatus for illuminating biological substrates for the purpose of viewing and detecting patterns of absorbance and fluorescence on said biological substrate. In particular, the present invention is directed to the use of light emitting diodes (LEDs) as light sources in a multiple wavelength transillumination system.  
       BACKGROUND OF THE INVENTION  
       [0003] The separation of proteins, nucleic acids and other biological materials by gel electrophoresis on polyacrylamide or agarose matrices is a standard technique in molecular biology (Westermeier, R. and Barnes, N., Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations 3rd edition, Vch Verlagsgesellschaft Mbh, 2001). A common method for analyzing these gels after electrophoresis is to immerse the gels in a solution containing a dye that binds to all or some of the separated materials. The gels are then destained to remove any unbound dye and the gel is placed on a transilluminator for viewing. The transilluminator emits light of a specific wavelength that is absorbed by the dye. Depending on the dye, it may or may not re-emit light at a longer wavelength (i.e., fluorescence). If the dye absorbs light but does not fluoresce, the stained material appears dark against a background of transmitted light (Sasse, J., and Gallagher, S. R., Current Protocols in Molecular Biology Unit 10.6, Ausubel, F. A., et al., eds., Wiley-Interscience, 1991). If the dye fluoresces and an optical filter is used to block the source light and to pass the emitted light, the stained material appears light against a dark background (Nucleic Acid Detection, TCO167, Molecular Probes Inc. 2000; Tools For Proteomics, TC0158-2, Molecular Probes Inc. 2002). These patterns can be detected and documented using a variety of techniques including photography using a camera with film and, more recently, imaging using a CCD camera.  
       [0004] Transilluminators used in the art to visualize dyes that absorb or fluoresce are described in a number of patents. The term “transilluminator” as used herein means a device which generates light and allows the light to pass through a diffuser or filter onto which a material has been placed and permits viewing of the material and any pattern generated in or on the material by the action of the light passing through or impinging on the material that is visible to the viewer or detector. Transilluminators are typically comprised of an open enclosure that houses a light source and a diffuser or filter that covers the light source and transmits light emitted by the source. A gel is placed on the diffuser or filter and light impinges on the gel. A detector or viewer is then used to record the pattern of absorbance or fluorescence caused by the interaction of the light and the dye used to stain the material in the gel. A variety of filters may be used to modify the light emitted by the light source or the dyes in order to enhance detection or viewing. Transilluminators that use a high intensity ultra-violet (UV) light source primarily for viewing nucleic acid gels stained with fluorescent dyes are described in U.S. Pat. Nos. 5,327,195, 4,657,655, and 5,347,342. A multiple wavelength UV transilluminator that contains three different lamps that cover the short, mid and long UV wavelengths is described in U.S. Pat. No. 5,387,801. A transilluminator capable of both UV and visible light illumination using interchangeable lamps is described in U.S. Pat. No. 4,071,883. Alternatively, a screen that can be placed on a UV transilluminator to convert UV to visible light is described in U.S. Pat. No. 5,998,789. More recently, a transilluminator for fluorometric detection using visible light generated from fluorescent lamps that emit in the blue spectrum is described in U.S. Pat. Nos. 6,198,107 and 6,512,236.  
       [0005] Historically, high intensity UV transilluminators were designed and developed to view nucleic acid gels stained with fluorescent dyes such as ethidium bromide. UV light for viewing gels has two major disadvantages: 1) exposure of humans viewing gels to intense UV light is hazardous and requires protection to eyes and skin to avoid damage and 2) exposure of biomolecules in gels to intense UV light can induce damage or adduct formation that can irreversibly alter the structure and function of the molecules making further study difficult.  
       [0006] In addition, not all fluorescent dyes, especially those used to stain proteins, absorb optimally in the UV region (Haugland, R., Handbook of Fluorescent Probes and Research Products, Ninth Edition, Molecular Probes, Inc., 2002). A transilluminator for fluorometric detection using visible light generated from fluorescent lamps filtered to emit light in the blue spectrum is described in U.S. Pat. Nos. 6,198,107 and 6,512,236. However, this device is limited to a specific set of fluorescent dyes that absorb in the blue region.  
       [0007] With the advent of proteomics, the most widely adopted method for studying proteins is two-dimensional gel electrophoresis (2DE). Proteins separated by 2DE are visualized by a variety of staining methods using visible dyes such as silver and Coomassie Blue and fluorescent dyes such as SYPRO Ruby and the CyDyes used for fluorescence 2D difference gel electrophoresis (2D DIGE).  
       SUMMARY OF THE INVENTION  
       [0008] In accordance with an embodiment of the present invention, a transilluminator for 2DE protein gels that is capable of viewing gels stained by any of a number of methods is provided. In particular, embodiments of the present invention provide a multi-wavelength transilluminator and methods for viewing and detecting patterns of light scattering, absorbance and fluorescence for a variety of staining technologies within a single device. The light source may be an array of high intensity narrow emission band light emitting diodes (LEDs) matched to the absorbance spectra of each dye type. The transilluminator includes a light source containing multiple LEDs emitting light at various wavelengths optimized for detecting specific dyes and an optical filter to ensure that the only light reaching the detector or viewer is light produced by fluorescence of the dyes. The optical filter is optional for detecting or viewing absorbance. The different LEDs can be selected in any combination during illumination and their intensity is adjustable using pulse width modulation (PWM) over a range from 0-100%.  
       [0009] In accordance with another embodiment of the present invention, a multiple wavelength transillumination system using LEDs as light sources is provided for the viewing and detection of patterns of absorbance and fluorescence. Different LEDs emitting different wavelengths of light are combined in the same device to form a multiple wavelength transillumination system. Different numbers of LEDs are combined to form transilluminators of different sizes. Uniform surface light illumination is achieved by:  
       [0010] placing a diffuser at an optimal distance from the LEDs, selecting LEDs with a sufficiently large angle of illumination and brightness,  
       [0011] spacing LEDs at a sufficient density and in an optimal pattern, and  
       [0012] properly adjusting the intensity of illumination.  
       [0013] In accordance with the present invention, a multiple wavelength transillumination system comprises:  
       [0014] 1) a light source consisting of LEDs capable of producing light in any combination of the following:  
       [0015] a) of the excitation type for commonly used fluorescent dyes (i.e., fluorophors) used to stain biomolecules,  
       [0016] b) of the type absorbed by commonly used colorimetric dyes (i.e., chromophors) used to stain biomolecules, and  
       [0017] c) of the type scattered by commonly used particulate dyes used to stain biomolecules.  
       [0018] 2) a diffuser placed between the light source and the dyes being viewed or detected;  
       [0019] 3) optional optical filter(s) placed between said fluorescent dyes and a viewer or light detector which filter is capable of transmitting light emitted by the fluorescent dyes and of preventing transmission of light from said light source of said excitation type, to form a viewable image of the pattern of fluorescent dyes. In some embodiments, the optical filter may be adapted to be placed over the human eye or may to be attached to the lens of an optical scanner, charge coupled device or camera.  
       [0020] The devices and methods of this invention are especially useful when the user requires viewing, detecting, comparing and imaging of the spatial arrangements of multiple chromophors and fluorophors either contained within the same matrix or contained in different matrices such as 1D and 2D protein and DNA electrophoresis gels, thin-layer chromatography plates (TLC), gel blots, chromatography fractions, arrays, biochips, and other analytical or preparative substrates.  
       [0021] The system of this invention may be incorporated into an integrated device such as a 2D gel processing system, gel documentation system, horizontal or vertical gel electrophoresis unit, scanner, imager or other device in which viewing or detection of absorbance, fluorescence and light scattering is required.  
       [0022] Devices of this invention use LEDs as light sources rather than ultraviolet, incandescent and fluorescent lamps of the types described in the U.S. Pat. Nos. 4,071,883, 4,657,655, 5,327,195, 5,347,342, 5,387,801, 6,198,107 and 6,512,236. Embodiments of the present invention use multiple high intensity, narrow band LEDs with large angles of illumination that permit viewing of several different chromophors and fluorophors. In these embodiments the wavelength(s) and intensity(ies) are selectable either mechanically, electronically or through software. Direct viewing may be accomplished using the human eye or viewing and recording may be accomplished using an imaging device such as a film camera using both conventional photography or a CCD camera as part of a digital imaging system. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0023]FIG. 1 is a perspective view of a transillumination device in accordance with an embodiment of the present invention;  
     [0024]FIG. 2 is an exploded view of the device of FIG. 1;  
     [0025]FIG. 3 is a functional block diagram of a transilluminator in accordance with an embodiment of the present invention;  
     [0026]FIG. 4 illustrates an LED circuit diagram comprised of two independent LED circuits in accordance with an embodiment of the present invention;  
     [0027]FIG. 5 illustrates an arrangement of LEDs on a printed circuit board in accordance with an embodiment of the present invention;  
     [0028]FIG. 6 illustrates an arrangement of LEDs on a printed circuit board in accordance with another embodiment of the present invention;  
     [0029]FIG. 7 illustrates a transillumination system in accordance with an embodiment of the present invention; and  
     [0030]FIG. 8 is a flow chart illustrating the operation of a device in accordance with an embodiment of the present invention.  
    
    
     DETAILED DESCRIPTION  
     [0031] Many dyes fluoresce light within the visible spectrum when illuminated by ultraviolet or visible light. Other dyes absorb or scatter light within the visible spectrum when illuminated by visible light. However, prior to the present invention, there have not been transilluminators capable of viewing or detecting a broad range of dyes within a single device. This is because transilluminators for viewing fluorophors place optical filters on either side of the material to which a fluorophor is bound to narrow the excitation band impinging on the material and to narrow the emission band passing to the detector. This allows the emitted light from the fluorophor to be detected but limits the fluorophors that can be detected since only a single pair of narrow excitation and emission wavelength bands are available. Transilluminators for viewing chromophors that absorb or scatter light use unfiltered white or broad-band visible light which is unsuitable for viewing fluorescent dyes.  
     [0032] The present invention does not require an excitation filter between the light source and the material to which a fluorophor is bound to narrow the excitation band impinging on the material being viewed. Rather it uses high intensity LEDs that emit a narrow band light suitable for direct illumination of the fluorophor. By placing a variety of narrow band LEDs in the same transilluminator and constructing a circuit whereby LEDs of one type can be controlled independently of those of another type, it is possible to construct a transilluminator that can emit a variety of different ultraviolet and visible excitation wavelengths as well as white or broad-band visible light. Furthermore, the circuit can include the capability to adjust intensity and to combine the various wavelengths. The circuit also can be designed so that these adjustments are made manually by a user or under software control. By placing a variety of emission filters between the material being viewed and the detector, many excitation/emission pairs can be supported. In addition, chromophors that absorb or scatter light can also be viewed with the same device.  
     [0033]FIG. 1 illustrates the composition of a transilluminator or transillumination device  100  in accordance with an embodiment of the present invention. In general, the transillumination device  100  includes a number of output or light source arrays  104  (e.g., light source arrays  104   a  and  104   b ) comprising one or more LEDs  112  that are mounted on a printed circuit board  116  and placed in an enclosure  120 . The LEDs  112  may comprise high intensity LEDs having wide angles of illumination (e.g., greater than about 120°). A diffuser  124  is mounted proximate to a side of the printed circuit board  116  from which light from the LEDs  112  is emitted, and a bottom plate  128  may be mounted on the other side of the printed circuit board  116 . The diffuser  124 , the pattern and density of the LEDs  112  mounted on the printed circuit board  116 , the illumination angle of the LEDs  112  and the distance between the diffuser  124  and the printed circuit board  116  all contribute to the uniformity of illumination.  
     [0034] With reference now to FIG. 2, the transillumination device  100  of FIG. 1 is shown in an exploded view. In particular, FIG. 2 illustrates the relationship between the diff-user  124 , enclosure  120 , printed circuit board  116 , and bottom plate  128 .  
     [0035] With reference now to FIG. 3, a transillumination system  300  including a transillumination device  100  in accordance with an embodiment of the present invention is illustrated in functional block diagram form. In general, the transilluminator  100  includes a control  304 , a first light source array  104   a , a second light source array  104   b , and a third light source array  104   c . In addition, the transillumination device  100  includes a diffuser  124 .  
     [0036] The control  304  may comprise a switch for selectively operating the associated light source arrays  104 , for example where the transillumination device  100  is under the manual control of an operator. Alternatively or in addition, the control  304  may comprise a programmable device executing instructions regarding the operation of the light source arrays  104 . In addition to providing manual or automated switching capabilities, the control  304  may be operated to vary the intensity of the light produced by the light source arrays  104 . For example, in accordance with an embodiment of the present invention, the control  304  provides a pulse width modulated signal to a light source array or arrays  104  being operated. As can be appreciated by one of skill in the art, by varying the duty cycle of a signal provided to a light source array  104 , the intensity of the light produced by the light source array  104  can be varied. For example, a pulse width modulated signal that was on for 50% of the time in a given time segment would result in a reduced intensity of the light output by a given light source array  104  as compared to a control signal in which the signal to the light source array  104  was on continuously. The ability to modulate the intensity of light output by a light source array  104  is particularly useful in connection with normalizing light received at the detector  316  between different light source arrays  104  and/or samples  308 . As can be appreciated by one of skill in the art, the control  304  may control a separate or integrated amplifier for providing power to the light source arrays  104 .  
     [0037] Each light source array  104  comprises one or more LEDs  112 . In accordance with an embodiment of the present invention, each light source array  104  comprises LEDs  112  that output light at a wavelength or within a range of wavelengths that is different from the light output by LEDs  112  of another light source array  104 . Accordingly, the transilluminator  100  can be operated to provide light at a selected wavelength, without requiring the use of an excitation filter between a light source and a sample or the changing of such an excitation filter. In accordance with another embodiment of the present invention, two or more light source arrays  104  may comprise LEDs  112  that output light at the same wavelength or range of wavelengths. Light source arrays  104  so configured may then be selectively operated to vary the intensity of light at the wavelength or range of wavelengths produced by the included LEDs  112 . The provision of multiple light source arrays  104  having LEDs at the same wavelength or range of wavelengths to vary intensity may be combined with the modulation of the control signal provided to the LEDs  112 .  
     [0038] Light produced by the light source arrays  104  is passed through the diffuser  124 . The diffuser  124  functions to diffuse the light, thereby providing substantially even illumination across the operating surface of the transilluminator device  100 . In accordance with an embodiment of the present invention, the diffuser  124  is formed from polypropylene.  
     [0039] The sample  308  is positioned on or adjacent the diffuser  124 , such that the light produced by a light source array  104  and passed through the diffuser  124  is incident upon the sample  308 . As can be appreciated by one of skill in the art, a sample  308  may comprise a biological substrate. Dye that has been bound to biological material on the biological substrate may then be viewed by light from the transillumination device  100  that is passed through or impinges on the material. Light comprising wavelengths resulting from the fluorescence of material being viewed, or light scattered by the material, may be selectively viewed by positioning an emission filter  312  between the sample  308  and the detector  316 . The detector  316  may comprise any device capable of detecting the fluorescence, scattering, or absorption of light by the material being visualized. Accordingly, examples of a detector  316  include a human eye, alone or in combination with a microscope, a photosensor device, or imaging device, including an optical scanner or a camera, including a film camera or a charge coupled device (CCD) camera.  
     [0040]FIG. 4 is a schematic diagram of a light source circuit  400  of a device in accordance with an embodiment of the present invention. In particular, FIG. 4 illustrates that a transillumination device  100  in accordance with embodiments of the present invention may comprise multiple circuits to allow light having different properties to be produced. For example, in FIG. 4, a transillumination device  100 , including a light source circuit  400  that has a first light source array  104   a  comprising a first circuit  404   a  containing LEDs  112   a  that emit light within a first wavelength range and a second light source array  104   b  comprising a second circuit  404   b  containing LEDs  112   b  that emit light within a second wavelength range is schematically depicted. As shown in FIG. 4, the first circuit  404   a  may be operated to illuminate the LEDs  112   a  of the first light source array  104   a  and the second circuit  404   b  may be operated to illuminate the LEDs  112   b  of the second light source array  104   b  independently of one another. Accordingly, an embodiment having a light source circuit  400  as illustrated in FIG. 4 may be operated to produce light within either or both of first and second wavelength ranges.  
     [0041]FIG. 5 illustrates an arrangement of LEDs  112  on a printed circuit board  116  in accordance with an embodiment of the present invention. In the embodiment of FIG. 5, rows of LEDs  112   a  included in a first light source array  104   a  alternate with rows of LEDs  112   b  included in a second light source array  104   b . The interleaving of LEDs  112   a associated with a first fight source array  104   a  with LEDs  112   b  associated with a second light source array  104   b  provides a transilluminator in which samples can be evenly illuminated by either the first  104   a  or second  104   b  light source arrays.  
     [0042] With reference now to FIG. 6, an arrangement of LEDs  112  on a printed circuit board  116  in accordance with another embodiment of the present invention is illustrated. In the embodiment of FIG. 6, the individual LEDs  112   a  and  112   b  of the first  104   a  and second  104   b  light source arrays respectively are interleaved. As with the embodiment illustrated in connection with FIG. 5, the embodiment illustrated in FIG. 6 provides even illumination of samples by either the first light source array  104   a  or the second light source array  104   b.    
     [0043] In order to optimize the uniformity of illumination provided by a light source array  104 , it may be necessary to use still other arrangements of LEDs  112 . In particular, the number of different types of LEDs  112  used by a light source array or arrays  104 , and the angle and intensity of illumination of the individual types of LEDs  112  may require the use of different LED  112  layouts. Optimal layouts may involve different geometric patterns as well as different numbers of LEDs  112  of each type. In accordance with additional embodiments of the present invention, transilluminators  100  of various sizes can be created by combining 1, 2, 3 . . . n printed circuit boards  116  to make a single light source array or combination of arrays  104 .  
     [0044] As can be appreciated by one of skill in the art, a transillumination device  100  may include more than two light source arrays  104  (as depicted in FIGS. 1 and 4- 6 ) or three light source arrays  104  (as depicted in FIG. 3). In particular, in connection with a transilluminator  100  capable of providing excitation light at more than two or three wavelengths or wavelength ranges, additional light source arrays and associated LEDs  112  may be included. For example, in accordance with an embodiment of the present invention, a first light source array  104   a  comprising LEDs  112  that output light at a first wavelength is combined with a second light source array  104   b  comprising LEDs  112  that output light at a second wavelength, a third light source array  104  comprising LEDs  112  that output light at a third wavelength and an n th  light source array  104  comprising LEDs  112  that output light at an n th  wavelength. By providing light source arrays  104  comprising LEDs  112  that are capable of outputting light at different wavelengths, it can be appreciated that different dyes within a sample  308  or in different samples  308  can be observed, particularly when a selected filter  312  is placed between a sample  308  and an observation device or detector  316 .  
     [0045] To demonstrate the efficacy of a transillumination system  300  including a transillumination device  100  in accordance with an embodiment of the present invention, a SYPRO Ruby stained 2D protein gel was transilluminated. A fluorescent staining pattern of the fluorophor dye bound to proteins in the gel was observed. In accordance with an embodiment of the present invention, the illumination was achieved using 470 nm Super Blue LEDs  112   a  provided as part of a first light source array  104   a  associated with a first circuit  404   a . A CCD camera (the detector  316 ) was fitted with a Red Additive 590 nm Long Pass emission filter  312  to capture an image. In addition, an image of a Coomassie Blue stained 2D protein gel transilluminated by a device  100  of this invention showing the colorimeteric staining pattern of the chromophor dye bound to proteins in the gel was obtained. An image of a Coomassie Blue stained 1D protein gel transilluminated by a device of this invention showing the colorimeteric staining pattern of the chromophor dye bound to proteins in the gel was also obtained. In accordance with an embodiment of the present invention, the transillumination device  100  used in the present example also contains white incandescent light LEDs  112   b  included as part of a second light source array  104   b  associated with a second circuit  404   b  for viewing protein gels stained with the chromophors silver or Coomassie Blue. The two types of LEDs  112   a - b  are arranged in the interleaved printed circuit board layout illustrated in FIG. 6. The emission filter  312  can remain in place when viewing these chromophors. The transillumination system  300  of the present example comprises a device that automatically images gels placed on the transilluminator as shown in FIG. 6. White or blue LED selection and intensity is adjustable through software control implemented as part of the control  304 .  
     [0046]FIG. 7 shows a transillumination device  100  integrated into an automated gel processing system or transillumination system  300  in accordance with an embodiment of the present invention. The transillumination system  300  includes a detector  316  comprising a downward looking CCD camera. The CCD camera  316  can be selectively fitted with an emission filter  312 . A carrier  704  allows the CCD camera  316  to be moved to a desired position over a platform  708 . The platform  708  may be transparent and/or may have an aperture or window to allow illumination of a sample or samples  308 . The platform  708  may also be configured to receive a tray  712  to which one or more biological substrates or slides comprising samples  308  may be attached. The tray  712  may be transparent and/or may be provided with a window or aperture to allow illumination of a sample or samples  308 .  
     [0047] To view or detect fluorescence, the light source array or arrays  104  are comprised of LEDs  112  selected for emission peaks close to the excitation peaks of the dyes being measured and an emission filters  312  is placed between the sample  308  comprising the dye and the viewer or detector  316  while the substrate or sample  308  is illuminated by the transillumination device  100 . The emission filter  312  is chosen to ensure that only the light of wavelengths emitted by the dye is passed to the viewer or detector  316 . A different emission filter  312  may be used for different LED/dye combinations. By selecting LEDs  112  of a sufficiently narrow wavelength band, there is no need for an excitation filter in the described device. This also makes the selection of the type of emission filter  312  less critical. The good separation between excitation and emission wavelengths achievable using LED illumination produces images with extraordinarily large signal to noise ratios. The light source array or arrays  104  for viewing or detecting fluorescence may include LEDs  112  chosen to excite dyes with excitation peaks in the ultra-violet and/or visible spectrum.  
     [0048] To view or detect absorbance, the light source array or arrays  104  are comprised of LEDs  112  that emit wavelengths of light over all or part of the absorbance range of the dyes being measured. Incandescent white LEDs  112  may be used as well as single color LEDs  112  or multiple single color LEDs  112 . Since the absorbance range of most dyes is broad, the selection of LEDs  112  for viewing absorbance is less critical than for fluorescence measurements. Ideally, the LEDs  112  emit light near the peak of absorbance for a specific dye. In many cases, the same LEDs  112  can be used for both fluorescence and absorbance measurements.  
     [0049] To view or detect light scattered by particles attached to biomolecules as stains, the light source array or arrays  104  are comprised of LEDs  112  that emit wavelengths of light scattered by the particles being measured. Incandescent white LEDs  112  may be used as well as single color LEDs  112  or multiple single color LEDs  112 . Since the light scatter range of most particles is broad, the selection of LEDs for use in connection with light scatter measurements is less critical than for fluorescence measurements. In many cases, the same LEDs can be used for fluorescence, absorbance and light scatter measurements.  
     [0050] The dyes used in connection with samples  308  illuminated by the transillumination device  100  in accordance with an embodiment of the present invention may be any chemicals, stains, dyes, chromophors, fluorophors, colloidal silver, colloidal gold, nanoparticles or other substances bound to and used to visualize a pattern, structure, substrate or substance known or readily available to those skilled in the art, and are preferably used in the form of dyes bound to or in a biological sample. The dyes may be used to detect and quantify any desired substance to which they can be attached or into which they can be incorporated (e.g. proteins, nucleic acids, carbohydrates, fats, scaffolds, supramolecular structures, cells, tissues and organisms). Dyes may also be an intrinsic part of an organism or substance to be visualized or detected (i.e., occurs naturally rather than being artificially stained with an exogenously added dye).  
     [0051] With reference now to FIG. 8, a flow chart illustrating a method for selectively viewing dyes within a sample  308  in accordance with an embodiment of the present invention is illustrated. Initially, at step  800 , a sample of biological material is stained with a dye. As can be appreciated by one of skill in the art, the dye may selectively bind to particular materials within the sample of biological material. As can also be appreciated by one of skill in the art, a number of different dyes may be used in connection with a single sample of biological material. The staining process may include destaining to remove any unbound dye.  
     [0052] At step  804 , the sample  308  is placed on the platform  708 , such that light emitted by the transillumination device  100  is incident upon the sample  308 . At step  808 , the wavelength of light required in order to observe or view a desired dye or aspect of the desired dye, and the pass band of an emission filter  312  required to view that dye or aspect of the dye are determined. The emission filter  312  is then positioned in front of the detector  316  (step  812 ), and the light source array  104  having LEDs  112  that produce light at the required wavelength is operated (step  816 ). As described elsewhere herein, the selection of light source wavelength and emission filter pass band allows fluorescence, absorption, or scattering by material within the sample  308  to be viewed or detected.  
     [0053] At step  820 , a determination is made as to whether the intensity of the light output by the selected LEDs  112  is appropriate. For example, when a number of different dyes within a sample are viewed or detected, the intensity of the image produced may vary. Accordingly, it may be desirable to normalize the intensity of the image, for example in order to facilitate a comparison of images of the different dyes. If the intensity of the light is not appropriate, the intensity of the light output may be varied, for example by providing a pulse width modulated control signal to the selected light source array  104  of LEDs  112  (step  824 ). After adjusting the intensity of the light output, or if no adjustment is required, the process proceeds to step  828 .  
     [0054] At step  828 , an image of the sample, and in particular of the dye or aspect of the dye being viewed or detected, is created. For example, a film photograph or digital image may be made of the illuminated sample  308 . Alternatively, the sample  308  may be viewed by a human eye directly or through a microscope. In one aspect of the present invention, the detector  316  may obtain a complete image of the sample  308  at one time. In particular, because a transillumination device  100  in accordance with the present invention is capable of illuminating the entire area of a sample  308  simultaneously, there is no need to build an image through rastering or other techniques.  
     [0055] At step  832 , a determination is made as to whether any additional dye within a sample  308  is to be viewed. If additional dyes are to be viewed, the process returns to step  808 , at which the wavelength of the light required to view the desired dye and the pass band of the emission filter  312  are determined. After positioning the emission filter  312  in front of the detector  316  (step  816 ), the light source array  104  having LEDs  112  that provide light at the required wavelength may be operated (step  820 ). It should be noted that operation of a transillumination device  100  such that light at a second wavelength or range of wavelengths that is different from a first wavelength or range of wavelengths does not require changing an emission filter. Rather, a different light source array  104  of LEDs  112  capable of producing light at the required wavelength is selected. In addition, it should be noted that no excitation filter is used. Instead, the proper wavelength of excitation light is obtained by the use of LEDs that output light at the required wavelength or range of wavelengths. If no additional dyes are to be viewed, the process ends (step  836 ).  
     [0056] Tables 1 and 2 below provide examples of dyes used in protein studies, their excitation and emission ranges, and the corresponding LEDs and filters required to view them in accordance with embodiments of the present invention.  
                           TABLE 1                                      Excitation (nm)   Emission (nm)                                 Dye   Range   Peak   Range   Peak               SYPRO Ruby   370-530 nm   470 nm   550-720 nm   610 nm           250-340 nm   290 nm   550-720 nm   610 nm       SYPRO Tangerine   380-560 nm   470 nm   560-750 nm   645 nm       SYPRO Orange   400-530 nm   470 nm   520-650 nm   550 nm       SYPRO Red   450-610 nm   530 nm   550-700 nm   630 nm       Cy2   450-520 nm   485 nm   490-570 nm   515 nm       Cy3   480-570 nm   550 nm   550-630 nm   570 nm       Cy3B   na   558 nm   560-640 nm   572/585 nm       Cy5   580-680 nm   650 nm   640-710 nm   670 nm       Cy5.5   na   675 nm   660-730 nm   694 nm       Silver   na   na   na   na       Coomassie Blue   na   na   na   na                  
 
     [0057]                       TABLE 2                       Dye   LED   Emission Filter                  SYPRO Ruby   470 nm Super Blue   Red Additive               590 nm Long Pass       SYPRO Tangerine   470 nm Super Blue   Red Additive               590 nm Long Pass       SYPRO Orange   470 nm Super Blue   Green Additive               490-580 nm               Band Pass       SYPRO Red   525 nm InGaN Super Green   Red Additive           λΔ36   590 nm Long Pass       Cy2   470 nm Super Blue   520 nm λΔ10           505 nm λΔ10   Band Pass       Cy3   502 nm Blue Green   568 nm λΔ10           λΔ30   Band Pass           502 nm Blue Green   580 nm λΔ10           λΔ30   Band Pass           525 nm InGaN Super Green   600 nm λΔ40           λΔ36   Band Pass       Cy3B   502 nm Blue Green   568 nm λΔ10           λΔ30   Band Pass           502 nm Blue Green   580 nm λΔ10           λΔ30   Band Pass           525 nm InGaN Super Green   600 nm λΔ40           λΔ36   Band Pass       Cy5   612 nm Orange   671 nm λΔ10           λΔ17   Band Pass               676 nm λΔ10               Band Pass               650 nm               Long Pass       Cy5.5   650 nm Ultra Red   700 nm λΔ40           λΔ20   Band Pass               694 nm λΔ10               Band Pass       Silver   470 nm + 525 nm   None       Coomassie Blue   525 nm + 612 nm   None                    
     [0058] In an exemplary embodiment of the present invention, a transilluminator  100  is comprised of the following:  
     [0059] 1) A light source circuit  400  containing three light source arrays  104 , each light source array  104  having LEDs  112  of one of the following types:  
     [0060] a) 470 nm Super Blue  
     [0061] b) 525 nm InGaN Super Green  
     [0062] c) 612 nm Orange  
     [0063] 2) Four emission filters  312 :  
                                                      a) Red Additive   590 nm Long Pass           b) Green Additive   490-580 nm Band Pass           c) 515 nm Narrow   Band Pass           d) 568 nm Narrow   Band Pass                      
 
     [0064] Such a transillumination device  100  can be used to view most, if not all, of the dyes listed in Tables 1 and 2. Other configurations optimized for other dye sets are possible.  
     [0065] One feature of this exemplary embodiment is the ability to image fluorescence  2 D differential electrophoresis gels (2D DIGE). 2D DIGE uses molecular weight- and charge-matched, spectrally resolvable dyes (e.g., Cy3(b) and Cy5) to label two different protein samples prior to 2D electrophoresis. By way of illustration, one protein sample may be isolated from cells treated in one way and the other protein sample isolated from cells treated in another way. One protein sample is labeled with a first dye and the other with a second dye. The samples are mixed and resolved on a single gel. The gel is imaged using two different excitation/emission pairs to view the pattern of fluorescence for each dye independently. Then the two images are aligned and the differences evaluated. By integrating such an embodiment into a gel imaging system, such as a transillumination system  300 , it is possible under software control to automatically image the Cy3(b) stained proteins of a sample  308  using the 525 nm InGaN Super Green/568 nm Narrow Band Pass pair and then automatically switch to the 612 nm Orange/Red Additive 590 nm Long Pass pair to image the Cy5 stained proteins of that sample  308 .  
     [0066] Based on the examples provided and the embodiments described, it should be understood that the multiwavelength transillumination system  300  as specifically described herein could be altered without deviating from its fundamental nature. For example, different LED light sources  112  and sets and types of filters  312  could be substituted for those exemplified and described herein, so long as the light reaching the light detector  316  contains sufficient information to allow viewing of an image of the pattern of absorbance, light scattering and fluorescence produced by the dyes being illuminated. As an additional example, the LEDs  112  could be positioned so that the light produced by the LEDs  112  impinged on a sample  308  from the side of the sample  308  facing the detector  316 . For instance, the LEDs  112  could be arranged in a ring surrounding the detector  316 . In accordance with the present invention, certain embodiments may allow detection and quantification of the amount of absorbance, light scattering and fluorescence produced by the dyes being illuminated. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced in ways other than as specifically described herein.  
     [0067] The foregoing discussion of the invention has been presented for purposes of illustration and description. Further, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, within the skill and knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain the best mode presently known of practicing the invention and to enable others skilled in the art to utilize the invention in such or in other embodiments and with various modifications required by their particular application or use of the invention. It is intended that the appended claims be construed to include the alternative embodiments to the extent permitted by the prior art.