Abstract:
The present invention relates to apparatus, systems, and methods for analyzing biological samples. In particular, the invention provides systems and methods for analyzing samples in a light microscope. The invention allows microscopic position sensing and focusing. The invention includes the use of at least two coordinated beams of light, one of which operates to determine the position of the other. In a preferred embodiment, the system is a microscope having total internal reflection optics installed. Systems of the invention can also be constructed from a standard microscope configured with a total internal reflection objective.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]     This application claims priority to U.S. provisional patent application Ser. No. 60/589,170, filed on Jul. 19, 2004, the disclosure of which is incorporated herein by reference in its entirety. This application also incorporates herein by reference a U.S. patent application filed of even date herewith and identified by Attorney Docket No. HLI-002B. 
     
    
     TECHNICAL FIELD  
       [0002]     The invention relates generally to apparatus, methods, and systems for handling and analyzing microfluidic volumes and related biological materials. In particular, the invention relates to optical equipment, such as lighting systems, for analyzing biological samples.  
       BACKGROUND  
       [0003]     Generally, systems for analyzing a sample in a flow cell are pressure driven fluidic systems using pressure pumps. Pressure driven fluidics systems have several disadvantages. One disadvantage is that pressure driven systems require the sample vessel to be sealably engaged to the flow cell assembly. This makes removal of the flow cell more complicated, because removal of the flow cell can produce hazardous aerosols. Pressure systems are also known to develop system leaks due to the pressure and may require frequent replacement of lines and valves. Additionally, pressure driven systems can introduce contaminants into the sample. Another disadvantage of pushing fluid through the system is that air can become trapped in the system or air bubbles can be introduced into the sample. Introduction of air into the pump can cause cavitation resulting in shock to the system. Moreover, in pressure driven systems, it is difficult to adequately purge the lines after each sample has been tested. This can result in residual material being left in the system when the next test is performed. Also, purging the system using air pressure tends to cause bubbling or foaming in the samples, which may introduce inaccuracies to the analysis.  
         [0004]     The prior art vacuum driven systems that have been used to analyze samples in a flow cell also have disadvantages. In these prior art systems, a vacuum pump is directly connected to the flow cell. Again, the use of a pump can cause air bubbles to be introduced into the sample and air trapped in the pump transmits shock to the system. Additionally, the continuous on and off cycle of the pump can result in uneven passage of a sample through the flow cell. Prior art vacuum systems are also generally suited for passing multi-cell samples through the flow cell. Having a pump directly connected to the flow cell can negatively impact single-cell samples, in part, because of the shock transmitted to the system.  
         [0005]     In analyzing microfluidic volumes and related biological materials using a light source, it is desirable for the light source to hit the sample in such a way that results in total internal reflection fluorescence (“TIRF”). TIRF is an optical phenomenon that occurs when light propagating in a dense medium, such as glass, meets an interface with a less dense medium such as water. If the light meets the surface at a small angle, some of the light passes through the interface (is refracted) and some is reflected back into the dense medium. At a certain angle, known as the critical angle, all of the light is refracted. However, some of the energy of the beam still propogates a short distance into the less dense medium, generating an evanescent wave. The evanescent wave only penetrates about 100 nm into the medium. If this energy is not absorbed, it passes back into the dense medium. However, if a flourophore molecule is within the evanescent wave, it can absorb photons and be excited. The excited fluorophores can be observed using, for example, an intensified CCD camera. Accurately maintaining the critical angle to obtain TIRF in a dynamic system is difficult.  
       SUMMARY OF THE INVENTION  
       [0006]     The invention provides systems and methods for analyzing samples in a light microscope. The invention allows microscopic position sensing and focusing. The invention comprises the use of at least two coordinated beams of light, one of which operates to determine the position of the other. In a preferred embodiment, the system is a microscope having total internal reflection optics installed. Systems of the invention can also be constructed from a standard microscope configured with a total internal reflection objective.  
         [0007]     In one aspect, the invention relates to a system including a first light source for analyzing a sample of interest and a second light source. The first light source defines a first optical path that intersects a sample of interest and the second light source operates with the first light source for determining a position of the first optical path.  
         [0008]     In various embodiments of the foregoing aspect, the first light source and the second light source operate simultaneously. The second light source may define a second optical path at least partially coaxial with the first optical path. In one embodiment, the second light source is directed to a position sensor for sensing an angle of reflection of the first optical path relative to the sample of interest. The position of the first optical path can be adjusted to vary the angle of reflection in response to a signal from the position sensor. The position of the first optical path can be adjusted to obtain substantially total internal reflection of the first light source relative to the sample of interest.  
         [0009]     Additionally, the first light source can have a wavelength from about 390 nm to about 1550 nm. In one embodiment, the second light source is infrared light. The first light source and/or the second light source can be a laser, a light emitting diode, or a lamp. In one embodiment, the system includes an imaging device for imaging the sample of interest. Further, the system can include a third light source for analyzing the sample of interest. The third light source can define a third optical path at least partially coaxial with the first optical path. The first light source and the third light source can be operated simultaneously. The second light source may be used to continuously monitor the position of the first optical path. In one application, the light system can be adapted for use in a single molecule sequencing system.  
         [0010]     In another aspect, the invention relates to a method of substantially maintaining total internal reflection for a sample of interest. The method includes the steps of providing a first beam of light for intersecting with the sample of interest, providing a second beam of light for determining a position of the first beam of light, directing the second beam of light onto a position sensor, and adjusting the position of the first beam of light in response to a signal from the position sensor to vary an angle of reflection of the first beam of light with respect to the sample of interest to substantially maintain total internal reflection.  
         [0011]     In various embodiments, the first beam of light is at least partially coaxial with the second beam of light. The first beam of light is for analyzing the sample of interest. In one embodiment, the first light source has a wavelength from about 390 nm to about 1550 nm. The second light source may be infrared light. The method may also include the steps of continuously monitoring the position of the first beam of light and adjusting the angle of reflection in response thereto to substantially maintain total internal reflection.  
         [0012]     In another aspect, the invention relates to a system for analyzing a sample. The system includes a flow cell, a passive vacuum source for pulling a volume through the flow cell, a lighting system for illuminating the sample in the flow cell, and an optical instrument for viewing the sample in the flow cell. The lighting system can be of the type described hereinabove. In one embodiment, the volume includes the sample or agents for reacting with the sample, which may be predisposed on or within the flow cell. Alternatively or additionally, the sample may adhere to or come to rest within the flow cell while the volume passes therethrough. In one embodiment, the volume and/or sample is moved through the flow cell by gravity. For example, the head pressure on the volume within an inlet to the flow cell is sufficient to move the volume through the flow cell.  
         [0013]     In various embodiments of the foregoing aspect, the system includes a stage for receiving the flow cell, where the stage is movable in at least one direction. In one embodiment, the stage is movable in two orthogonal directions. The system may also include an image capture device for capturing an image of the sample. The image capture device can be a charge coupled device (CCD), a complementary metal oxide semiconductor device (CMOS), a charge injection device (CID), or a video camera. Additionally, the system could include a processor for collecting and processing data generated by the system, storage for storing the data, and means for displaying at least one of the data and the sample.  
         [0014]     In another aspect, the invention relates to an apparatus for handling microfluidic volumes, such as biological samples for analysis. The apparatus can include the aforementioned passive vacuum source and flow cell. The microfluidic volume is pulled through the flow cell by the passive vacuum source. In one embodiment, the passive vacuum source includes a pump, a pump driver, such as an electric motor, and a reservoir. The pump can be connected to the reservoir and then operated to evacuate the reservoir, thereby creating a vacuum within the reservoir. In one embodiment, the vacuum pressure is from about 1″ Hg to about 29″ Hg. The vacuum pressure can be adjusted to vary the speed at which the microfluidic volume passes through the flow cell.  
         [0015]     In various embodiments of the foregoing aspects, the apparatus/system can be used for single molecule detection. In one embodiment, the flow cell includes a surface for receiving a nucleotide. For example, the flow cell can include a bound nucleotide and a primer bound to the nucleotide and/or the flow cell. In particular, the flow cell can include a slide and a coverslip, where the nucleotide and/or the primer are bound to at least one of the slide and the coverslip. Additionally, the flow cell can include a channel for pulling the microfluidic volume therethrough.  
         [0016]     In some embodiments of the foregoing aspects, the ratio of a volume of the reservoir and the microfluidic volume is between about 1,000:1 and about 2,000,000:1, or between about 50,000:1 and about 1,000,000:1, or about 200,000:1. Further, the apparatus can include valving disposed between the various components thereof. For example, the apparatus can include a valve disposed between the vacuum source, for example the reservoir, and the flow cell, wherein the valve includes an open position to connect the flow cell to the vacuum source and a closed position to isolate the flow cell from the vacuum source. The apparatus can also include a vacuum pressure indicator connected to the reservoir. Moreover, the apparatus can further include optical equipment for analyzing material within the flow cell after exposure to the microfluidic volume.  
         [0017]     In another aspect, the invention relates to a method of detecting single molecules. The method includes the steps of depositing a sample comprising single molecules into a flow cell, the flow cell treated to identify specific molecules; applying a vacuum to the flow cell; pulling the sample through a channel defined by the flow cell; and viewing the flow cell after exposure to the sample to identify the molecules exposed to the flow cell.  
         [0018]     In another aspect, the invention relates to a method of detecting single molecules. The method includes the steps of providing a flow cell that defines a channel that is treated to identify specific molecules, applying a vacuum to the channel to pull a sample through the channel, the sample comprising single molecules, and viewing the sample in the channel to identify the single molecules.  
         [0019]     Various embodiments of the foregoing methods include the step of removing the vacuum from the flow cell after pulling the sample through the channel. The step of applying a vacuum can include exposing the flow cell to a passive vacuum source. In various embodiments, the sample includes a microfluidic volume including nucleotides. Additionally, the flow cell can include at least one of a slide and a coverslip treated to bind with a specific nucleotide. Further, the step of viewing the flow cell can include illuminating the flow cell with a lighting system, such as that described hereinabove. The step of viewing the flow cell can also include using an image capture device. In one embodiment, a processor is used to control the operation of the method. The processor can be used for collecting and processing data generated during the method. The method can further include the step of displaying at least one of the flow cell and the data.  
         [0020]     In another embodiment, single nucleotide detection is accomplished by attaching template nucleic acids to a flow cell in the presence of a primer for template-dependent nucleic acid synthesis. Using a device according to the invention, a vacuum is created across the flow cell for introduction of reagents for template-dependent nucleic acid synthesis. For example, once template/primer pairs are bound to the surface of the flow cell, reagents comprising labeled or unlabeled nucleotides and a polymerase to catalyze nucleotide addition are added via an entry port. The vacuum is switched on and the reagents are exposed to the flow cell and then exit via an exit port to the reservoir. After a wash step, complementary nucleotides added to primer are detected. Preferably, reagent nucleotides are labeled with, for example, a fluorescent dye. Such dyes are observed using light microscopy. For example, cyanine dyes (cyanine-3 or cyanine-5) are useful for optical detection of incorporated nucleotides. Using optically-detectable labels, nucleic acid sequencing is conducted on a single molecule level. This means that individual template nucleic acids are positioned on the flow cell such that each is individually optically resolvable. The location of the templates is determined by, for example, the use of dye-labeled primers that hybridize to individual templates. Labeled nucleotides are flowed across the flow channel using the mechanisms described herein under conditions that allow complementary nucleotide addition to the primer. Once incorporated, the label is detected by excitation of the dye at the appropriate wavelength and by using an emission filter for detection of the emission spectrum. Emissions that occur at a location known to contain a template indicate incorporation of the labeled base at that position. By conducting these steps multiple times, a sequence is completed. Single molecule sequencing techniques are described in Braslavsky, et al., PNAS (USA), 100: 3960-3964 (2003) and copending U.S. patent application Ser. No. 09/707,737, each of which is incorporated by reference herein.  
         [0021]     In another aspect, the invention relates to a flow cell for analyzing single molecules, such as nucleotides. The flow cell includes a slide, a coverslip, and a gasket disposed between the slide and the coverslip. The slide, the coverslip, and the gasket define a microfluidic channel for passing single molecules under vacuum. In various embodiments, the flow cell includes a nucleotide bound to the slide and/or the coverslip. In addition, the flow cell can include a primer bound to at least one of the nucleotide, the slide, and the coverslip. In one embodiment, the slide includes a plurality of nucleotides bound thereto.  
         [0022]     In another aspect, the invention relates to a slide for use with a flow cell. The slide can include at least one nucleotide bound to a surface of the slide. The slide can be disposed within the flow cell. The slide can further include a primer bound to at least one of the slide and the nucleotide. In addition, the slide can include a plurality of nucleotides bound thereto.  
         [0023]     In another aspect, the invention relates to a coverslip for use with a flow cell. The coverslip includes at least one nucleotide bound to a surface of the coverslip. The coverslip can be disposed within the flow cell. The coverslip can further comprise a primer bound to at least one of the coverslip and the nucleotide. In one embodiment, the coverslip includes a plurality of nucleotides bound thereto.  
         [0024]     These and other objects, along with advantages and features of the present invention herein disclosed, will become apparent through reference to the following description, the accompanying drawings, and the claims. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0025]     In the drawings, like reference characters generally refer to the same parts throughout the different views. Also, the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present invention are described with reference to the following drawings, in which:  
         [0026]      FIG. 1  is a schematic representation of one embodiment of an apparatus for handling microfluidic volumes in accordance with the invention;  
         [0027]      FIG. 2  is a schematic representation of an alternative embodiment of an apparatus for handling microfluidic volumes in accordance with the invention;  
         [0028]      FIG. 3  is a schematic representation of another alternative embodiment of an apparatus for handling microfluidic volumes in accordance with the invention;  
         [0029]      FIG. 4A  is a pictorial representation of one possible configuration of the apparatus of  FIG. 1 ;  
         [0030]      FIG. 4B  is a pictorial representation of a portion of the apparatus of  FIG. 4A ;  
         [0031]      FIG. 5  is a plan view of a portion of the apparatus of  FIG. 4A ;  
         [0032]      FIG. 6  is a side view of a portion of the apparatus of  FIG. 4A ;  
         [0033]      FIG. 7A  is a block diagram of a system in accordance with one embodiment of the invention;  
         [0034]      FIG. 7B  is a pictorial representation of the system of  FIG. 7A ;  
         [0035]      FIG. 8  is a flow chart depicting one mode of operation of a method of handling microfluidic volumes in accordance with the invention;  
         [0036]      FIG. 9  is a plan view of a flow cell in accordance with one embodiment of the invention;  
         [0037]      FIG. 10  is a cross-sectional view of the flow cell of  FIG. 9  taken at line  10 - 10 ;  
         [0038]      FIG. 11  is an exploded view of the flow cell of  FIG. 9 ; and  
         [0039]      FIGS. 12A and 12B  are schematic representations of a lighting system in accordance with one embodiment of the invention. 
     
    
     DESCRIPTION  
       [0040]     Embodiments of the present invention are described below. It is, however, expressly noted that the present invention is not limited to these embodiments, but rather the intention is that modifications that are apparent to the person skilled in the art are also included. For example, many of the following embodiments are described with reference to pulling microfluidic volumes through a flow cell, however, the present invention can also be applied to pulling fluids through other types of analytical equipment, such as, for example, flow cytometers and chemical analyzers. Further, the apparatus can be used as part of a system for detecting single molecules by, for example, optical detection of single nucleotides.  
         [0041]     In one embodiment, the apparatus  10  includes a vacuum source  12 , an isolation valve  20 , and a flow cell  30 . In the embodiment depicted in  FIG. 1 , the vacuum source  12  is passive and includes a vacuum pump  14 , a drive motor  16 , and a reservoir  18 . Alternatively, the vacuum source  12  could be non-passive, where the vacuum pump  14  is directly connected to the flow cell (see, for example,  FIG. 3 ). In one embodiment, the vacuum pump  14  is a compact rotary vane type pump; however, the pump size and type will be selected to suit the particular application. For example, the pump could be a piston, gear, or diaphragm type pump. Further, the pump size will depend on the operating parameters of the apparatus  10 , for example, the larger the pump capacity, the quicker the pump  14  will evacuate the reservoir  18 . The drive motor  16  in one embodiment is a 12 volt DC electric motor; however, the motor size and type will be selected to suit the particular application. For example, larger flows may require a larger pump, which in turn may require a larger motor. Further, the pump  14  can be uni- or bi-directional and can be coupled to the motor  14  directly or via a flexible coupling or other means known to one of skill in the art. In a particular embodiment, the pump  14  and motor  16  are supplied as an assembly, such as model no. 50200 available from Thomas Pumps and Compressors of Shebogan, Wis.  
         [0042]     The reservoir  18  in one embodiment is a four liter bottle, such as Nalgene® model no. 2125-4000 available from Nalge Nunc International of Rochester, N.Y. The reservoir size will be selected to suit a particular application and, as will be discussed in greater detail below, is typically substantially larger than the microfluidic volume to be pulled by the vacuum source  12 . In addition, the reservoir material can be a metal, a polymer, glass, or combinations thereof. In particular, the reservoir material should be compatible with the microfluidic volume  32 . Also, the reservoir  18  should be capable of withstanding the pressures to which the reservoir  18  is exposed. For example, the reservoir  18  should be able hold a vacuum with minimal leakage and without collapsing.  
         [0043]     The apparatus  10  shown in  FIG. 1  includes three valves,  20 A,  20 B,  20 C (collectively  20 ). The valves  20  shown are two position, three connection type solenoid valves, such as model no. LHDA1233115H available from the Lee Co. of Westbrook, Conn. The solenoids, which actuate the valves, are energized by 12 volt DC; however, other voltages can be used and the valves can be actuated hydraulically, pneumatically, or manually. Additionally, the valve type and configuration can be selected to suit a particular application. For example, the valves can be two position, two connection or two position, four connection.  
         [0044]     The first valve  20 A is located between the reservoir  18  and the pump  14 . In the unactuated state, the valve  20 A isolates the reservoir  18  from the pump  14 . The pump inlet  40  is connected to the atmosphere, while the reservoir outlet  42  is closed. Alternatively, the pump inlet  40  could be closed. When the first valve  20 A is actuated, for example by energizing the solenoid, the valve  20 A changes position, thereby connecting the pump inlet  40  to the reservoir outlet  42  and allowing the pump  14  (when running) to pull a vacuum on the reservoir  18 . In one embodiment, the vacuum pressure is between about 1″ Hg and about 29″ Hg, preferably between about 2″ Hg and 15″ Hg, and more preferably between about 5″ Hg and about 6″ Hg; however, the vacuum pressure can be varied to suit a particular application. Generally, the greater the vacuum pressure, the faster the microfluidic volume  32  will be pulled through the flow cell. In some cases, a fast flow is desirable to reduce the amount of residue left within the flow cell  30  from the microfluidic volume  32 .  
         [0045]     The second valve  20 B is located between the reservoir  18  and the flow cell  30 . In the unactuated state, the valve  20 B isolates the reservoir  18  from the flow cell  30 . The reservoir inlet  44  is closed, while the flow cell outlet  46  is connected to the atmosphere. Alternatively, the flow cell outlet  46  could also be closed. When the second valve  20 B is actuated, the valve  20 B changes position, thereby connecting the flow cell outlet  44  to the reservoir inlet  46 , which results in the vacuum within the reservoir  18  pulling the volume of material  32  through the flow cell  30 . The vacuum pressure within the reservoir  18  determines the speed at which the volume  32  is pulled through the flow cell  30 .  
         [0046]     Optionally, a third valve  20 C, as shown in  FIG. 1 , is connected to the reservoir  18  and is used to vent the reservoir  18 . The optional third valve  20 C could be located at a different location on the apparatus  10  to perform a different function. Alternatively or additionally, multiple valves  20  can be used in conjunction with multiple flow cells  30 . For example, the apparatus  10  can include ten flow cells  30 , or other analytical equipment, each connected in series with a valve  20  and the reservoir  18  (see, for example,  FIG. 2 ).  
         [0047]     The flow cell  30  is coupled to the vacuum source  12 , as described above. Multiple flow cells  30 , or other analytical equipment, can be connected to the vacuum source  12  either in series or in parallel (see, for example,  FIG. 2 ). In one embodiment, the flow cell  30  is a Focht Chamber System (model no. FCS2) available from Bioptechs of Butler, Pa. Alternatively, a customized flow cell system may be used. The flow cell  430  depicted in  FIGS. 9-11  is a customized flow cell and will be described in greater detail with respect to  FIGS. 9-11   
         [0048]     Further depicted in  FIG. 1  is a pipette  50  for introducing the microfluidic volume  32  to the apparatus  10 ; however, other types of vessels can be used for introducing the volume  32  to the apparatus  10 . For example, a cuvette or beaker could be used. The pipette  50  is positioned directly over the flow cell inlet  48 . In one embodiment, the microfluidic volume  32  includes single molecules for use in sequencing deoxyribonucleic acid (DNA). In one embodiment, the pipette  50  can manually or automatically dispense individual microfluidic volumes in the range of about 2 microliters (μl) to about 2 milliliters (ml), preferably about 10 μl to about 100 μl, and more preferably about 20 μl. Further, the pipette  50  can be handled robotically to, for example, position the pipette  50  relative to the flow cell inlet  48 , receive and mix materials within the pipette  50 , and/or dispense precisely the microfluidic volume  32  based on time and/or volume.  
         [0049]     The apparatus  10  further includes a pressure indicator  60 , such as model no. DPG1000B-30INHGVAC available from Omega Engineering, Inc. of Stamford, Conn. The indicator  60  is used to measure the vacuum pressure within the reservoir  18 ; however, additional indicators can be used to measure the pressure at other locations in the apparatus  10 , for example, the flow cell outlet  46 . The indicator  60  can be a pressure gauge, a pressure transducer, and/or pressure switch, with or without a readout. For example, the pressure transducer could include a digital readout of the actual vacuum pressure within the reservoir  18  and/or the pressure switch can activate an alarm if the pressure within the reservoir  18  reaches a threshold value.  
         [0050]     The apparatus  10  depicted in  FIG. 1  also includes an optional controller  70 . The controller  70  includes the electronic controls for operating, for example, the vacuum source  12  and valves  20  by, for example, a computer  68  and related software. The apparatus  10  can send and receive data directly or via the controller  70  to the computer  68 . The computer  68  can be a conventional computer system including a processor, hard drive, RAM, a video monitor, and a keyboard, as may be found in a laboratory setting. The computer  68  can interact with the controller  70  to store and process data as necessary to operate the apparatus  10 . Alternatively or additionally, the controller  70  can include an internal data processor. Alternatively, the apparatus  10  can be controlled manually. The controller  70  shown is a switch and sense type controller available from Measurement Computing Corporation of Middleboro, Mass. The exact controller configuration will be selected based on, for example, the number of inputs and outputs required and the type of equipment to be controlled. In one embodiment, the controller  70  can include the logic for cycling the pump  14  and motor  16  on and off and actuating the valves  20  based on predetermined time intervals and/or in response to signals from sensors. The controller can also supply the necessary power to the various components of the apparatus  10 .  
         [0051]      FIG. 2  depicts schematically an alternative embodiment of an apparatus  110  in accordance with the invention. The apparatus  110  is similar to the apparatus  10  described hereinabove with respect to  FIG. 1 ; however, the apparatus  110  shown in  FIG. 2  includes multiple flow cells  130 ,  130 ′,  130 ″ and corresponding second valves  120 B,  120 B′,  120 B″ arranged in a parallel configuration. As described above, the apparatus  110  includes a passive vacuum system  112  including a pump  114 , a motor  116 , and a reservoir  118 ; a first valve  120 A; a pressure indicator  160 ; and a controller  170 .  
         [0052]     The multiple flow cells  130 ,  130 ′,  130 ″ and the corresponding second valves  120 B,  120 B′,  120 B″ are arranged in parallel to facilitate running multiple operations either simultaneously or sequentially. For example, the user can run three different operations without having to change set-ups between operations. The large ΔV between the reservoir  118  and the microfluidic volumes  32 ,  32 ′,  32 ″ facilitates multiple operations without any degradation in performance. Alternatively or additionally, the flow cells  130  could be arranged serially; however, serially arranged flow cells  130  would have to be operated simultaneously and may impact the adjacent flow cell(s)  130 .  
         [0053]      FIG. 3  depicts schematically another alternative embodiment of an apparatus  210  in accordance with the invention. The apparatus  210  is similar to the apparatus  10 ,  110  described hereinabove with respect to  FIGS. 1 and 2 ; however, the apparatus  210  shown in  FIG. 3  does not include a reservoir. The apparatus  210  includes a non-passive vacuum system  212  including a pump  214  and a motor  216 , where the pump  214  is directly connected to the flow cell  230  via a single valve  220 . The apparatus  210  further includes a pressure indicator  260  located between the pump inlet  240  and the flow cell outlet  246 , and a controller  270 .  
         [0054]      FIG. 4A  is a pictorial representation of one possible configuration of the apparatus  10  depicted schematically in  FIG. 1 . The vacuum system  12 , valves  20 , and indicator  60  are mounted on a breadboard  72 ; the reservoir  18  is free-standing adjacent to the breadboard  72 ; and the flow cell  30  is disposed on a microscope type stage  52  adjacent to the breadboard  72 . The breadboard  72  is mounted on top of the controller  70  via stand-offs  74  and screws  76  located at the four corners of the breadboard  72 . Also mounted on the breadboard  72  are push-buttons  56  for operating the valves  20 , and the electrical and fluidic connections for the various components.  
         [0055]     As shown in  FIG. 4A , the apparatus  10  uses tubing  54  to connect the various components, for example, the pump  14  and reservoir  18 . In one embodiment, the tubing  54  is capillary type tubing, which can be obtained from, for example, Polymicro Technologies, LLC of Phoenix, Ariz. Alternatively or additionally, conventional polymer tubing can be used, for example, ⅛″ outside diameter nylon, such as Nylotube® available from New Age Industries, Inc. of Southampton, Pa. The size, type, and material of the tubing can be selected to suit a particular application. For example, metallic tubing may be undesirable for biological materials and the size of the tubing  54  should be selected based on the flow parameters of the microfluidic volumes. For example, the inside diameter of the tubing  54  should be sufficient to prevent turbulent flow of the microfluidic volume therethrough.  
         [0056]     Moreover, the apparatus  10  can include various optical components, such as a microscope objective, a camera, and multiple light sources for optically analyzing the contents of the microfluidic volume  32  and/or the operation of the apparatus  10 . Additionally, the flow cell  30  can be located on a microscope type stage  52  for optical viewing by the user. In one embodiment, the stage  52  can be moved in the X, Y, and/or Z directions to position the flow cell  30  relative to the optical components. In an alternative embodiment, the flow cell  30  is secured within a stationary fixture. Alternatively or additionally, the optical components can be movable in the X, Y, and/or Z directions. The apparatus  10  can also include additional sensors for monitoring various operations of the apparatus  10 . For example, the apparatus  10  could include an optical sensor for monitoring the level of the microfluidic volume  32  within the flow cell inlet  48 .  
         [0057]      FIG. 4B  is a pictorial representation of a portion of the apparatus  10  shown in  FIG. 4A . Specifically,  FIG. 4B  depicts an enlarged view of the flow cell  30  from the side opposite that shown in  FIG. 4A . The flow cell inlet  48  is shown open and unobstructed. In operation, there would be a pipette located above the flow cell inlet  48 . The pipette would contain and dispense the microfluidic volumes to be pulled through the flow cell  30 . Shown above the flow cell inlet  48  is a camera  80  that can be used to display an image of the flow cell inlet  48  and the fluid flow therethrough to the user on, for example, an optional video monitor. Alternatively or additionally, the image can be used in conjunction with a sensor to send a signal to the controller  70  to, for example, close the second valve  20 B. The flow cell outlet  46  is shown with a fitting and capillary tubing running therefrom. The fitting  74  is a conventional type of fitting that can be used to connect the tubing to the flow cell outlet  46 , for example, a nut and ferrule type fitting. The tubing runs to the second valve  20 B (see  FIG. 4A ). Shown adjacent to the flow cell  30  is a heater  58  that can be used to heat the various components, for example the flow cell  30 , as needed to carry out a particular operation.  
         [0058]     The apparatus  10  will be further described with reference to  FIGS. 4A, 4B ,  5 , and  6 . The pump  14  and motor  16  are mounted to the breadboard  72  by a bracket  74 . The three valves  20  are also secured to the breadboard  72 . The pump  14  has two connections; the inlet  40  and an outlet  41 . The outlet  41  is open to the atmosphere, but could include an exhaust filter  24  ( FIG. 1 ) or be plumbed to a remote location. The inlet  40  is plumbed to an outlet  43  on the first valve  20 A via the tubing  54 . The inlet  45  of the first valve  20 A is than plumbed to the reservoir  18 . The connections between the pump  14 , valves  20 , and reservoir  18  are push type fittings, where the tubing  54  is pushed over the fittings and secured by friction and/or barbs. Other types of fittings are also contemplated and considered within the scope of the invention.  
         [0059]     An outlet  47  on the second valve  20 B is plumbed to the reservoir  18 . An inlet  49  on the second valve  20 B is plumbed to the flow cell  30 . The third valve  20 C is optional in the depicted configuration and is, therefore, not shown plumbed. The pressure indicator  60  includes an inlet  51  that is plumbed to the reservoir  18  to continuously monitor the vacuum pressure therein.  
         [0060]     Each of the valves  20  and the motor  16  include electrical connections  53 . The electrical connections  53  are wired to the controller  70  for connection to the necessary power source(s) and control logic. The push buttons  56 A,  56 B,  56 C,  56 D (collectively  56 ) also include electrical connections that are wired to the valves  20 , motor  16 , and controller  70 . The controller  70  includes an electrical connection  78  for connecting the controller  70  to the computer  68  (see  FIG. 1 ). The controller  70  may include an additional connection for connecting to an external power source. In one embodiment, the electrical connection  78  is a USB connection. Alternatively or additionally, the controller  70  could include an IEEE 1394 connection, such as the FIREWIRE® brand sold by Apple Computer, Inc. The controller  70  can further include a power switch and indicators, either alone or as part of a user interface.  
         [0061]     In the embodiment shown, the push buttons  56  are used to run the motor  16 , which drives the pump  14 , and to actuate the valves  20  by energizing the valve solenoids  21 . Specifically, the first push button  56 A, when pushed, energizes the motor  16 , thereby causing the pump  14  to pull a vacuum. The second push button  56 B, when pushed, energizes the first valve solenoid  21  A, thereby connecting the pump  14  to the reservoir  18 . When both push buttons  56 A,  56 B are pushed, the pump  14  evacuates the air out of the reservoir  18 , thereby creating a vacuum within the reservoir  18 . The third push button  56 C, when pushed, energizes the second valve solenoid  21 B, thereby connecting the reservoir  18  to the flow cell  30 . The fourth push button, when pushed, energizes the third valve solenoid  21  C, thereby actuating the third valve  20 C. The apparatus  10  can include additional valves and push buttons as required by the specific configuration. In addition, other types of switches could be used to operate the various components, as opposed to the push buttons shown. For example, toggle type switches could be used.  
         [0062]      FIG. 7A  depicts schematically an embodiment of a system  300  in accordance with the invention that includes an apparatus  310  and auxiliary components in accordance with the invention.  FIG. 7B  depicts one possible arrangement of the various components of the system. The auxiliary components include a lighting/optics module  320 , a microscope module  330 , and a computer module  340 . Generally, in one embodiment, the lighting/optics module  320  includes multiple light sources and filters to provide light to the microscope for viewing and analysis. The light is reflected onto, for example, a flow cell  312  seated on the microscope module  330  (see  FIG. 7B ). The light can be multiple wavelengths, for example, one wavelength for viewing and another wavelength for analysis. A particular lighting/optics module  600  is described with respect to  FIGS. 12A and 12B .  
         [0063]     The microscope module  330  includes hardware for holding the flow cell  312  and moving the microscope stage and an imaging device, such as a camera. In some embodiments, the microscope module  330  is a part of the apparatus  310 . The computer module  340  includes the memory and processors necessary for operating the various modules and a user interface for operating the system  300 . The modules communicate with one another as shown by the arrows in  FIG. 7A . For example, the computer module  340  may send a signal to the lighting/optics module  320  based on a user input to, for example, send a red light to the microscope module  330  to illuminate the flow cell. The computer module  340  can also send and receive signals from the microscope module  330  to change and monitor the position of the microscope stage or other operational parameters. Additionally, the computer module  340  can send and receive signals from the apparatus  310  to open and close valves.  
         [0064]     As shown in  FIG. 7B , the various components of the system  300  are mounted on a laboratory bench  302  in close proximity to one another; however, the arrangement of the various components can vary to suit a particular application and/or environment. The microscope module  330  includes a stage  332  for positioning the flow cell  312  or other item to be analyzed, a camera  334 , and optics  336 . Generally, a microscope, such as model no. TE2000 from Nikon Instruments, Inc. of Melville, N.Y., is suitable for use with the system  300 ; however, the type of microscope used can be selected based on the particular application and the nature of the sample to be analyzed.  
         [0065]     The computer module  340  includes a processor  342 , a video monitor  346 , and a user interface  344 , such as a keyboard and mouse for interacting with the system  300 . In one embodiment, the camera  334  sends images to the computer module  340  for analysis and/or display on the video monitor  346 . The lighting/optics module  320  of the system  300  includes an arrangement of light sources  342 ,  344  and filters  346  and mirrors  348  for conditioning the light emitted by the light sources  342 ,  344 . The arrangement of the components will vary to suit a particular application and/or environment. The lighting/optics module  320  supplies conditioned light to the microscope module  330  for the viewing and analysis of the sample disposed therein.  
         [0066]      FIG. 8  represents the basic operation  500  of an apparatus in accordance with one embodiment of the invention. Generally, a user monitors the vacuum condition within the reservoir (step  510 ). If, for example, the vacuum level is not within set limits, the user can increase the vacuum pressure within the reservoir by operating the vacuum pump (Steps  520 ,  530 ). Once the vacuum pressure is within the set limits, the user can deposit a sample (e.g., a microfluidic volume) into the flow cell inlet (Step  540 ). Subsequently, the user will open the flow cell outlet to the reservoir, thereby pulling the sample through the flow cell (Step  550 ). Once the user or the controller determines that the flow cell inlet is empty (Step  560 ), the connection between the flow cell outlet and the reservoir is closed (Step  570 ). The user and or controller will maintain the connection between the flow cell outlet and the reservoir open until the flow cell inlet is empty, as it is desirable to pull essentially all of the sample through the flow cell to prevent contaminating subsequent operations. If there are additional samples to be pulled through the flow cell (Step  580 ), the basic operation is repeated until there are no more samples, at which time the operation is ended (Step  590 ). Alternatively or additionally, the sample to be analyzed is contained within the flow cell, where the sample is exposed to the material or volume of material pulled through the flow cell, thereby causing a reaction or otherwise effecting the sample within the flow cell.  
         [0067]     More specifically, in operation, the user creates a vacuum in the reservoir  18  by, for example, operating the pump  14  and motor  16  and actuating the first valve  20 A isolating the pump  14  from the reservoir  18 . Once the desired vacuum is reached, for example about  6 ″ Hg, the first valve  20 A is deactuated and the pump  14  and motor  16  are stopped. Next, the pipette  50  deposits a microfluidic volume  32  within the flow cell inlet  48  and, subsequently, the second valve  20 B is actuated, thereby connecting the vacuum reservoir  18  to the flow cell outlet  46  and pulling the microfluidic volume  32  through the flow cell  30  and into the reservoir  18 , thus resulting in a transient exposure of the microfluidic volume and its contents to, for example, nucleotides that are held within the flow cell. Furthermore, the sample or volume can be driven through the flow cell by virtue of gravity, specifically the head of the volume held within the flow cell inlet or pipette. Once the microfluidic volume  32  leaves the flow cell inlet  48 , the second valve  20 B is closed, thereby removing the vacuum pressure from the flow cell  30 . Generally, the second valve  20 B should be open only long enough to pass the microfluidic volume  32  through the flow cell  30 . If the valve  20 B is open too long, air and bubbles can be pulled into the flow cell  30 ; if not open long enough, a portion of the volume  32  will remain in the flow cell  30 , which could contaminate subsequent operations. Subsequently, the sample can be viewed and analyzed as desired.  
         [0068]     In operation, it is desirable for the ratio of the reservoir volume  18  to the microfluidic volume  32  to be very large. For example, the ratio can be from about 1000:1 to about 2,000,000:1, preferably from about 50,000:1 to about 1,000,000:1, and more preferably about 200,000:1. In one embodiment, the reservoir  18  is about 4 liters (l) and the microfluidic volume is about 20 μl, thereby resulting in a ratio of about 200,000:1. The exact ratio will depend on, for example, the leakage rate of the reservoir, the size of the microfluidic volume, and the number of operations to be performed. A particularly large ratio results in the operation of the apparatus  10  being substantially unaffected by leakage and/or the number of microfluidic volumes  32  pulled through the flow cell  30 , because the reservoir volume under vacuum is so great relative to the volumes being absorbed by the reservoir, the change in volume is negligible. For example: 
 
P 1 V 1 =P 2 V 2 , where 
 
         [0069]     P 1 =the vacuum pressure within the reservoir prior to adding the microfluidic volume (ΔV);  
         [0070]     V 1 =the volume within the reservoir prior to adding ΔV;  
         [0071]     P 2 =the vacuum pressure within the reservoir after adding ΔV; and  
         [0072]     V 2 =the volume within the reservoir after adding ΔV.  
         [0000]     Because V 1  is so large relative to ΔV, V 1  is substantially equal to V 2 . Therefore, P 1  is substantially equal to P 2 .  
         [0073]     The valves  20 , pipette  50 , and pump  14  can be operated manually or automatically. For example, the second valve  20 B can be programmed to actuate (i.e., open) for “x” seconds after the pipette  50  deposits the volume  32  into the flow cell inlet  48  and deactuate (i.e., close) at the end of a set time period. In one embodiment, the time period can be adjusted to accommodate different volumes  32 . In an alternative embodiment, an optical sensor can be used to actuate and/or deactuate the second valve  20 B. For example, the second valve  20 B can be actuated after the optical sensor senses that the appropriate volume  32  has been deposited into the flow cell inlet  48  and deactuated after the sensor senses that the flow cell inlet  48  is empty. In one embodiment, the sensor(s) will send a signal to the controller  70 , which in turn outputs the appropriate response to the signal, e.g., deactuate the second valve  20 B. Additionally, the pressure sensor  60  can be used to control the first valve  20 A and the pump  14 . For example, if the pressure sensor  60  senses that the vacuum in the reservoir  18  has degraded below a threshold value, the controller  70  can turn on the pump  14  and motor  16  and actuate the first valve  20 A to increase the vacuum in the reservoir  18 .  
         [0074]      FIGS. 9, 10 , and  11  depict the customized flow cell  430 . The flow cell  430  is similar to the Focht Chamber System and includes a connection ring  436 , an upper gasket  438 , a slide  440 , a lower gasket  442 , a coverslip  444 , and a locking base  428 . Also shown is an optional heater  458 . The connection ring  436  sits on top of the various components and when seated and locked in the base  428  seals the components in place. It is desirable to operate the flow cell  430  by pulling a volume through under vacuum, as opposed to a pushing the volume through by positive pressure. Positively pressurizing the flow cell  430  may result in the slide  440  and/or coverslip  444  being bowed outwardly, contamination being trapped between the gaskets  438 ,  442  and the slide  440  and/or coverslip  444 , or otherwise compromising the integrity of the flow cell&#39;s structure. By using vacuum, the contact areas between the gaskets  438 ,  442  and the slide  440  and coverslip  444  are maintained, thereby eliminating the possibility of contamination collecting in those contact areas.  
         [0075]     The connection ring  436  houses the flow cell inlet  448  and the flow cell outlet  446 . In the embodiment shown, the inlet  448  and the outlet  446  are machined through the ring  436 . The inlet  448  is a conical shaped recess and the outlet  446  is a threaded connection for accepting a fitting. The conical shaped inlet  448  is as large as possible to facilitate viewing the flow of any microfluidic volumes deposited therein. The connection ring  436  also defines a viewing area  432  where the slide  440  and coverslip  444  are visible. Further, the connection ring  436  should be made of a material that is dimensional stable, compatible with the microfluidic volumes passed therethrough, and to which any substances within the microfluidic volumes will not stick. Such materials include, for example, polyetheretherketone, sold by PLC Corporation under the trademark PEEK®; polyoxymethylene, sold by DuPont under the trademark Delrin®; polytetrafluoroethylene, sold by DuPont under the trademark Teflon®; and ethlene-chlorotrifluorethylene, sold by Allied Chemical Corporation under the trademark Halar®.  
         [0076]     The upper gasket  438  provides the seal between the slide  440  and the connection ring  436 . In the embodiment shown, the upper gasket  438  has a thin annular shape; however, the size and shape of the upper gasket  438  will vary to suit a particular application. The lower gasket  442  provides the seal between the slide  440  and the coverslip  444 . In the embodiment shown, the lower gasket  442  covers a substantial portion of an upper surface of the coverslip  444 . In particular, the lower gasket  442 , along with a lower surface  441  of the slide  440 , and an upper surface  445  of the coverslip  444 , defines a flow channel  434  through which the microfluidic volumes travel. The size and shape of the flow channel  434  can be varied to suit a particular application. For example, the lower gasket  442  can be about 10 microns to about 3 millimeter (mm) thick, and can define an opening (flow channel  434 ) about 0.5 mm to about 5 mm wide, and the length of the opening can run substantially the entire width of the flow cell  430 . In one embodiment, the lower gasket  442  is about 50 microns thick and the flow channel  434  is about 1 mm wide by about 25 mm long. Alternatively, the microfluidic flow channel  434  could be etched in the slide  440  and/or the coverslip  444 .  
         [0077]     In operation, the microfluidic volume is deposited into the flow cell inlet  448  on the connection ring  436  and is pulled through the flow cell  430  under vacuum. The volume travels through the flow cell  430  as shown by the arrows in  FIG. 10 . Specifically, the volume travels downwardly through the connection ring  436  and through openings  439 B,  443 B in the upper gasket  438  and the slide  440 , and then into the flow channel  434  in the lower gasket  444 . The volume then travels through the flow channel  434  defined by the coverslip  444 , the slide  440 , and the lower gasket  442 . Once the volume reaches the opposing opening  443 A in the slide  440 , the volume is drawn upwardly through the openings  443 A,  439 A in the slide  440  and the upper gasket  438  and out the flow cell outlet  446  by the vacuum pressure within, for example, the reservoir. In various embodiments, the slide  440  and/or coverslip  444  can be treated to react with the microfluidic volume being pulled through the flow cell  430 . For example, a plurality of DNA strings can be adhered to the coverslip in the area corresponding to the flow channel  434  in the lower gasket  442 . Such an application is described in greater detail below.  
         [0078]     One application for an apparatus in accordance with the invention includes performing single molecule sequencing. In this application, the flow cell includes individual strands of DNA or RNA (the template) bound to, for example, the coverslip  444  of the flow cell  430  (see  FIGS. 9 and 10 ). The DNA or RNA can be bound to the coverslip by any known means for binding DNA or RNA to a surface using, for example, biotin-avidin interactions or other suitable attachment chemistries. A primer is added that hybridizes to a portion of the DNA or RNA bound in the flow cell.  
         [0079]     The coverslip or other components of the flow cell that are exposed to the flow path of the microfluidic volume can be produced and sold with specific oligonucleotides bound thereto. Further, the coverslip material can include glass, quartz, silicon, or other materials present in commonly-available nucleic acid array chips. The material can incorporate an epoxide surface or another suitably reactive material to facilitate binding of the DNA or RNA to the surface.  
         [0080]     In one embodiment, the DNA or RNA to be sequenced is immobilized on the slide or coverslip using a biotin/streptavidin linkage. Alternatively, immobilization can occur via the primer. For example, a biotinylated primer can be immobilized on the coverslip via streptavidin linked to biotin on the surface. Subsequent exposure of the immobilized primer to complementary DNA or RNA leads to sequence-specific hybridization with the DNA or RNA strand to be sequenced.  
         [0081]     Next, a microfluidic volume comprising a polymerase and a solution of nucleotides is pulled through the flow cell and exposed to the bound templates. Complementary nucleotides will be incorporated in the primer. Detectable labels are used to improve detection. Detection, however, can occur by detecting the indicia of nucleotide incorporation, for example, heat produced by the reaction or pyrophosphate production resulting from incorporation. By monitoring nucleotide incorporation over time, the user can thus determine the sequence of the exposed nucleotide at that position on the slide or coverslip. Because the apparatus permits parallel monitoring of a very large number of individually-resolvable single molecules, each at a separate position on the coverslip, a correspondingly large amount of sequence information can be collected at one time. Thus, computer systems are useful to monitor the observed label during the process and for handling the resulting sequence data. Depending on the nature of the DNA or RNA molecules sequenced, the apparatus can be used, for example, to identify nucleic acid sequence variations associated with disease; to select or monitor a course of treatment; or to monitor gene expression in an individual or in a population of individuals.  
         [0082]     In another embodiment, single nucleotide detection is accomplished by attaching template nucleic acids to a flow cell in the presence of a primer for template-dependent nucleic acid synthesis. Using a device according to the invention, a vacuum is created across the flow cell for introduction of reagents for template-dependent nucleic acid synthesis. For example, once template/primer pairs are bound to the surface of the flow cell, reagents comprising labeled or unlabeled nucleotides and a polymerase to catalyze nucleotide addition are added via the flow cell inlet. The vacuum is switched on and the reagents are exposed to the flow cell and then exit via the flow cell outlet to the reservoir. After a wash step, complementary nucleotides added to primer are detected. Preferably, reagent nucleotides are labeled with, for example, a fluorescent dye. Such dyes are observed using light microscopy. For example, cyanine dyes (cyanine-3 or cyanine-5) are useful for optical detection of incorporated nucleotides. Using optically-detectable labels, nucleic acid sequencing is conducted on a single molecule level. This means that individual template nucleic acids are positioned on the flow cell such that each is individually optically resolvable. The location of the templates is determined by, for example, the use of dye-labeled primers that hybridize to individual templates. Labeled nucleotides are flowed across the flow channel using the mechanisms described herein under conditions that allow complementary nucleotide addition to the primer. Once incorporated, the label is detected by excitation of the dye at the appropriate wavelength and by using an emission filter for detection of the emission spectrum. Emissions that occur at a location known to contain a template indicate incorporation of the labeled base at that position. By conducting these steps multiple times, a sequence is completed. Single molecule sequencing techniques are described in Braslavsky, et al., PNAS (USA), 100: 3960-3964 (2003) and copending U.S. patent application Ser. No. 09/707,737.  
         [0083]     A system for analyzing a sample in accordance with one embodiment of the invention includes a lighting system  600 . The lighting system  600 , as shown in  FIGS. 12A and 12B , may include a light source  602 , a primary filter  604 , a secondary filter  606 , a shutter  608 , a collimating lens  609 , a focusing lens  610 , and a power source  612 . A first portion of the lighting system  600 , shown in  FIG. 12A , includes three light sources  602 A,  602 B,  602 C (collectively  602 ). The lighting system  600 , however, may include only two light sources or additional light sources as needed. The light source  602  can include lasers, light emitting diodes, or lamps. In one embodiment, the first light source  602 A has a wavelength from about 390 nm to about 780 nm. In one embodiment, the first light source  602 A is a red laser. The second light source  602 B has a wavelength from about 936 nm to about 1340 nm. In one embodiment, the second light source  602 B is an infrared laser. The third light source  602 C has a wavelength from about 390 nm to about 780 nm. In one embodiment, the third light source  602 C is a green laser.  
         [0084]     The lighting system  600  shown in  FIG. 12A  also includes three primary filters  604 A,  604 B,  604 C (collectively  604 ). The primary filters  604  can include notch filters. The notch filters  604  are selected to transmit the desired wavelength and to block unwanted wavelengths emitted by each light source  602 . Additionally, the lighting system  600  shown in  FIG. 12A  includes three secondary filters  606 A,  606 B,  606 C (collectively  606 ). The secondary filters  606  can include dichroic filters. In one embodiment, the dichoric filters are placed at a 45’ angle relative to the light source  602 . With a dichroic filter positioned at a 45° angle relative to the light source  602 , a light source that would have been transmitted by the filter is still transmitted by the filter, but a light source that would have been blocked by the filter is reflected at a 90° angle. The lighting system  600  can also include shutter(s)  608  for blocking the light source(s)  602 . Additionally, the focusing lens  610  can be used for narrowing the beam emitted from the light source  602 , and the collimating lens  609  can be used for re-expanding and collimating the beam from the light source  602  to the desired diameter. In one embodiment, the three light sources  602 A,  602 B,  602 C are collimated to substantially the same diameter. It is desirable for the beams of the light sources  602  to be of substantially the same diameter and strength when they contact the sample of interest so that the field of illumination of the sample  620  is of equal size regardless of which light source  602  is used. Also, the lighting system  600  can include a power source  612  for providing power to the light sources  602 . The lighting system  600  can also include one or more mirrors for altering the optical path of the light sources as needed.  
         [0085]     The lighting source  602  is directed to a desired point. As shown in  FIG. 12B , the first light source  602 A can define a first optical path  630  that intersects a sample of interest  620 . The second light source  602 B can be used to determine the position of the first optical path  630 . Referring to  FIG. 12A , the first light source  602 A emits a beam of light of a desired wavelength in a desired optical path  630 . The beam of light passes through the focusing lens  610  that narrows the beam and then through the collimating lens  609  that re-expands and collimates the beam to a desired diameter. The beam of the first light source  602 A can be blocked by shutter  608  or allowed to pass through as desired. The beam of light passes through the notch filter  604 A, where only the light of the desired wavelength is permitted to pass through. The beam of light from the first light source  602 A then reflects off the first dichroic filter  606 A at a 90° angle to the angle of incidence. The beam of light from the first light source  602 A passes through the subsequent or downstream dichroic filters  606 B,  606 C in the desired optical path  630 .  
         [0086]     A second light source  602 B emits a beam of light of a desired wavelength. The beam of light then passes through notch filter  604 B, where only the light of the desired wavelength is allowed to pass through. The beam of light from the second light source  602 B then reflects off the dichroic filter  606 B at a 90° angle to the angle of incidence, such that the beam of the second light source  602 B is at least substantially coaxial (i.e., propogates along the same axis) with the optical path  630  of the beam of the first light source  602 A. The beams from the first light source  602 A and the second light source  602 B have substantially the same diameter. Both the beam from the first light source  602 A and the beam from the second light source  606 B pass through the third dichroic filter  606 C.  
         [0087]     A third light source  602 C, which may be used in addition to or as an alternative to the first light source  602 A, emits a beam of light of a desired wavelength. The beam of light passes through the focusing lens  610  that narrows the beam and then through the collimating lens  609  that re-expands and collimates the beam to the desired diameter. The beam can be blocked by the shutter  608  or allowed to pass through. The light then passes through the third notch filter  604 C where only the light of the desired wavelength is allowed to pass through. The beam of light from the third light source  602 C then reflects off the third dichroic filter  606 C at a 90° angle to the angle of incidence, such that the beam of the third light source  602 C is at least substantially coaxial with the first light source  602 A and/or the second light source  602 B. The beam of the third light source  602 C has substantially the same diameter as the beams from the first light source  602 A and second light source  602 B.  
         [0088]     Because the first light source  602 A and the third light source  602 C can be independently blocked, variations of which beams are directed to the desired position are possible. For example, the third light source  602 C can be blocked so that only the first light source  602 A and the second light source  602 B are directed to the desired point. Alternatively, all three light sources  602 A,  602 B,  602 C, can be directed to the desired point at the same time. In some embodiments, the lighting system can also include a neutral density filter  624  that is used to adjust the density of the light that is allowed to contact the sample  620 . For example, if the sample  620  is saturated with light, the neutral density filter  624  can be adjusted to reduce the strength of the light directed to the sample  620 . The neutral density filter  624  can be disposed along the optical path  630 .  
         [0089]     As shown in  FIG. 12B , the optical path  630  of the coaxial beam of the light source  602  is directed to a mirror  614  (or alternatively a dichroic filter). The optical path is reflected at a 90° angle to the angle of incidence towards a filter  616 . The beam from the first light source  602 A and/or the third light source  602 C reflects off filter  616  at a 90° angle to the angle of incidence towards the sample of interest  620 . The beam of the second light source  602 B is refracted by the filter  616  towards a position sensor  622  that senses the angle of reflection of the optical path  630  relative to the sample  620 .  
         [0090]     The information provided by the position sensor  622  could be used to adjust the angle θ at which the optical path  630  of the light source  602 A intersects the sample  620 . For example, the stage upon which the sample resides could be repositioned with respect to the optical path  630  and/or the orientation of the mirror  614  could be adjusted. Alternatively, the lighting system  600  could include a translator  618  that can be used to modify the angle of the optical path  630  of the light source  602 A towards the mirror  614 . The translator  618  can include a micrometer that is used to set the desired angle  0  of the optical path  630 .  
         [0091]     The desired optical path  630  is one that results in total internal reflection of the beam of the light source  602 A relative to the sample of interest  620 . The angle θ is the critical angle, and its value depends on the refractive indices of the media (θ=sin −1  (dense medium/less-dense medium). Thus the angle θ depends on the density of the glass (i.e., “dense medium”), the quality of the surface of the glass, and the density of the sample (i.e., “less-dense medium”).  
         [0092]     The position sensor  622  can be in communication with a computer, which can send a signal to automatically adjust the direction of the optical path  630  in response to a signal from the position sensor  622 . Alternatively, the position sensor  622  could have a read out that informs the user of the angle of reflection θ of the optical path  630 , which in turn could be manually adjusted. The angle θ of reflectance of the optical path  630  can be continuously monitored and adjusted as necessary to maintain the critical angle θ, as the system operates.  
         [0093]     When the light source  602  hits the sample  620  at the desired angle  0 , all of the light is reflected (i.e., there is total internal reflection). Some of the energy of the beam, however, still propagates a short distance into the less dense medium, generating an evanescent wave. A flourophore molecule attached to the sample of interest  620  absorbs photons of the evanescent wave and is excited. The excited fluorophores can be observed using, for example, an intensified CCD camera.  
         [0094]     The lighting system as illustrated in  FIGS. 12A and 12B , and as described above, is one possible arrangement of components of a lighting system in accordance with the invention. Other embodiments using different component arrangements, including different quantities and types of components such as filters and mirrors, are contemplated and considered within the scope of the invention. For example, multiple components can be used for conditioning the light source and adjusting the optical path or additional light sources could be used. Also, multiple sensors could be used to determine the angle of reflectance θ of the optical path  630 .  
         [0095]     Having described certain embodiments of the invention, it will be apparent to those of ordinary skill in the art that other embodiments incorporating the concepts disclosed herein may be used without departing from the spirit and scope of the invention. The described embodiments are to be considered in all respects as only illustrative and not restrictive.