Method and apparatus for detection of fluorescently labeled materials

What is claimed is:

1. An apparatus for detecting fluorescently marked regions on a first surface of a substrate, said apparatus comprising:

an excitation light source;

an optical train for directing an excitation light from said excitation light source at said substrate, and separating reflected excitation light from said first surface from fluoresced light from said first surface, said optical train comprising a spatial filter having a first lens and a second lens and a confocal pinhole located between said first lens and said second lens, a beam splitter cube, and a dichroic mirror for passing light having a wavelength of about said fluoresced light and reflecting light having a wavelength of about said excitation light, an optical lens and a microscope objective for directing said light at said substrate;

a focusing system for determining a focal plane of said excitation light passing through said optical train, said focusing means providing data for locating said first surface at said focal plane;

a detector for detecting said fluoresced light from said fluorescently marked regions in response to said light;

an x-y-z translation system for translating said substrate from a first position to a second position;

a flow cell mounted on said translation system, said flow cell comprising a mounting surface with a cavity therein, said mounting surface including a means for mounting said substrate thereon, and maintaining a sealed relationship with said substrate, whereby said first surface of said substrate is in fluid communication with said cavity, said cavity having an inlet and an outlet, and said inlet being connected to a pump for transferring materials into said cavity through said inlet and out of said cavity through said outlet; and

a storage system for storing a set of values representing an intensity of said fluoresced light as a function of the location on said substrate fluorescing said fluoresced light.

2. The apparatus as recited in claim 1, further comprising a video display for displaying said values representing the intensity of said fluoresced light as a function of location on said substrate.

3. The apparatus as recited in claim 1, wherein said focusing means comprises:

a photodiode for generating a voltage representing an intensity of said light reflected from said substrate;

a focusing lens for focusing said reflected excitation light from said substrate, from said optical train at said photodiode; and

a means for moving said substrate relative to a microscope objective until said intensity of said reflected excitation light focused at said photodiode from said substrate substantially reaches a maximum.

4. The apparatus as recited in claim 3, wherein a confocal pinhole is located between said focusing lens and said photodiode.

5. An apparatus as recited in claim 1, wherein said detecting means comprises:

a photomultiplier tube; and

a lens for focusing said fluoresced light separated by said optical train, at said photomultiplier tube.

6. An apparatus as recited in claim 5, wherein a confocal pinhole is located between said focusing lens and said photomultiplier tube.

7. An apparatus as recited in claim 5, wherein said photomultiplier tube is coupled to a means for collecting pulses generated by said photomultiplier tube in response to said fluoresced light, said means for collecting pulses being connected to a programmable computer for storing and analyzing said pulses.

8. An apparatus as recited in claim 1, further comprising means for controlling temperature in said flow cell, said means for controlling temperature including a recirculating bath device for circulating water through channels disposed in said flow cell.

9. A method for detecting fluorescently marked regions on a substrate, said method comprising the steps of:

immobilizing said substrate on a body, said body comprising a mounting surface having a cavity disposed therein, said substrate being immobilized on said mounting surface such that a probe array fabricated on said substrate is in fluid communication with said cavity, said cavity comprising a inlet and a outlet for flowing fluids into and through said cavity;

directing an excitation light from an excitation light source at said substrate;

auto-focusing said substrate in a focal plane of said excitation light;

exciting a first region of said substrate with said excitation light from said excitation light source, said excitation light source having a first wavelength;

detecting fluoresced light from said substrate in response to said excitation light, said fluoresced light having a second wavelength, said detecting comprising separating said light having a first wavelength from said light having a second wavelength and detecting said light having a second wavelength;

exciting a subsequent region on said substrate;

repeating steps of detecting and exciting a subsequent region until all regions of said substrate have been excited; and

processing and storing said fluoresced light to generate a 2-dimensional image of said substrate.

10. The method as recited in claim 9 wherein said body further comprises a temperature controller for controlling the temperature in said cavity.

11. The method as recited in claim 9, wherein said step of detecting comprises the steps of:

collecting said fluoresced light through optics; and

directing said fluoresced light from said optics onto a detector.

12. The method as recited in claim 9, wherein said step of exciting said subsequent region comprises the step of translating said substrate to allow said excitation light to excite said subsequent region.

13. The method as recited in claim 9, wherein said auto-focusing step comprises the steps of:

a) focusing a first surface of said substrate;

b) focusing a second surface of said substrate; and

c) finely focusing said second surface.

14. The method as recited in claim 13, wherein said step of focusing said first surface comprises the steps of:

directing said excitation light at said substrate, said excitation light being reflected by said substrate;

focusing said reflected excitation light from said substrate through a confocal pinhole;

detecting an amount of said reflected excitation light passing through said confocal pinhole, said confocal pinhole configured such that said amount of said reflected excitation light is at substantially a maximum when said first surface is located in substantially a focal plane of said excitation light;

moving said substrate closer to said excitation light and repeating the directing, focusing, detecting and moving steps until said amount of reflected excitation light passing through said confocal pinhole and detected in said detecting step has peaked.

15. The method as recited in claim 14, wherein said step of focusing said second surface comprises the steps of:

after focusing said first surface, first moving said substrate closer relative to said excitation light, a distance which said substrate is moved being equal to about half a thickness of said substrate;

directing said excitation light at said substrate, said excitation light being reflected by said substrate;

focusing said reflected excitation light through said confocal pinhole;

detecting said amount of reflected excitation light passing through said confocal pinhole;

second moving said substrate closer relative to said excitation light and repeating the second moving, directing, focusing, and detecting steps until said amount of reflected excitation light passing through said confocal pinhole and detected in said detecting step has peaked.

16. The method as recited in claim 15, wherein said step of finely focusing said second surface comprises the steps of:

directing said excitation light at said substrate;

focusing said reflected excitation light through said confocal pinhole;

detecting said amount of reflected excitation light passing through said confocal pinhole; and

moving said substrate farther relative to said excitation light and repeating the directing, focusing, detecting, determining, and moving steps until said amount of reflected excitation light passing through said confocal pinhole has reached a desired value.

17. The method as recited in claim 13, wherein said steps a-c are repeated for each corner of said substrate.

18. The method as recited in claim 17, further comprising the step of interpolating a focus position of each corner to determine said focus position of each region of said substrate.

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

The invention provides a method and associated apparatus for detecting and analyzing reactions of fluorescently marked materials on a single substrate surface.

Certain macromolecules are known to interact and bind to other molecules having a very specific three-dimensional spatial and electronic distribution. Any large molecule having such specificity can be considered a target. The various molecules that targets selectively bind to are known as probes.

Methods and devices for detecting fluorescently marked targets on devices are known. Generally, the devices includes a microscope and a monochromatic or polychromatic light source for directing light at a substrate. A photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate. A computer controls the movement of the x-y translation table and data collection. Such devices are discussed in, for example, U.S. Pat No. 5,143,854 (Pirrung et al.) incorporated herein by reference for all purposes. See also PCT WO 92/10092 also incorporated herein by reference for all purposes.

Light from the light source is focused at the substrate surface by manually adjusting the microscope. Manual adjustment is, on occasion, time consuming and inconvenient. Moreover, due to inherent imperfections present in the x-y translation table and substrate, there is a possibility that the substrate will be out of focus as it is moved from one region to another. As a result, the data collected may be misrepresented.

Also, temperature sometimes impact a chemical reaction between targets and probes. Generally, targets are more active or form stronger bonds at lower temperatures while the converse is true at higher temperatures. However, if the temperature is too low, the binding affinity of the target may become excessively strong, thus causing target to bind with complements (matches) as well as non-compliments (mismatches). Hence, the ability to control temperature may affect optimum binding between the targets and probes while minimizing mismatches.

In addition, the microscope detection devices are uneconomical to use. Typically, these devices incorporates the use of a microscope, and a multichannel scaler, both of which are costly.

From the above, it is apparent that an improved method and apparatus for detecting fluorescently labeled targets on a substrate is desired.

SUMMARY OF THE INVENTION

Methods and devices for the detection of fluorescently labeled targets on a substrate are disclosed. The detection method and devices utilize a substrate having a large variety of probes at known locations on its surface. The substrate, when placed in a confocal detection device, is exposed to fluorescently labeled targets that bind to one or more of the probes.

The confocal detection device includes a monochromatic or polychromatic light source, means for directing an excitation light from the light source at the substrate, means for focusing the light on the substrate, means for controlling temperature of the substrate during a reaction, means for detecting fluorescence emitted by the targets in response to the excitation light by directing the fluorescence through confocal pinholes, and means for identifying the region where the fluorescence originated. The means for controlling the temperature may include a temperature controlled fluid filled flow cell. The means for detecting the fluorescent emissions from the substrate, in some embodiments, include a photomultiplier tube. The means for focusing the excitation light to a point on the substrate and determining the region the fluorescence originated from may include an x-y-z translation table. Further, translation of the x-y-z table, temperature control and data collection are recorded and managed by an appropriately programmed digital computer.

In connection with one aspect of the invention, methods for analyzing the data collected by the fluorescent detection methods and devices are disclosed. Data analysis includes the steps of determining fluorescent intensity as a function of substrate position from the data collected; removing "outliers" (data deviating from a predetermined statistical distribution); and calculating the relative binding affinity of the targets from the remaining data. The resulting data are displayed as an image with the intensity in each region varying according to the binding affinity between targets and probes therein.

By using confocal optics, as well as focusing and temperature regulating techniques in conjunction with the data analysis methods, it is possible to quickly and accurately determine the relationship between structure and activity of certain molecules. Therefore, the potential for discovering novel probes with desirable pattern of specificity for biologically important targets is dramatically increased.

A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a detection system for locating fluorescent markers on the substrate;

FIG. 1b shows an alternative embodiment of a detection system for locating fluorescent markers on the substrate;

FIG. 1c shows another embodiment of a detection system for locating fluorescent markers on the substrate;

FIG. 2 is a flow chart illustrating the operation of the detection system;

FIGS. 3a and 3b show another flow chart illustrating the focusing step of the detection system;

FIGS. 4a and 4b show another flow chart illustrating the data acquisition step of the detection system;

FIG. 4c shows the relationship among the counters in the data acquisition board versus time.;

FIG. 5 is another flow chart illustrating the method of converting data representing photon counts as a function of position to data representing fluorescence intensity level as a function of position; and

FIGS. 6a and 6b show another flow chart illustrating the data analysis step .

DESCRIPTION OF THE PREFERRED EMBODIMENT

CONTENTS

I. Definitions

II. Details of One Embodiment of a Fluorescent Detection Device

III. Details of the Operation of a Fluorescent Detection Device

IV. Details of One Embodiment of Data Analysis to Determine Relative Binding Strength of Targets

V. Conclusion

I. Definitions

The following terms are intended to have the following general meanings as they are used herein:

1. Complementary: Refers to the topological compatibility or matching together of interacting surfaces of a probe molecule and its target. Thus, the target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.

2. Probe: A probe is a molecule that is recognized by a particular target. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

3. Target: A target is a molecule that has an affinity for a given probe. Targets may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended. A "Probe Target Pair" is formed when two macromolecules have combined through molecular recognition to form a complex.

II. Fluorescent Detection Device

FIG. 1a schematically illustrates a device used to detect fluorescently labeled targets on a substrate. Substrate 230 comprises a number of presynthesized probes on its surface 231. The substrate on which the sequences are formed may be composed from a wide range of material, either biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc. The substrate may have any convenient shape, such as a disc, square, sphere, circle, etc. The substrate is preferably flat but may take on a variety of alternative surface configurations. For example, the substrate may contain raised or depressed regions on which a sample is located. The substrate and its surface preferably form a rigid support on which the sample can be formed. The substrate and its surface are also chosen to provide appropriate light-absorbing characteristics. For instance, the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof. Other substrate materials will be readily apparent to those of skilled in the art upon review of this disclosure. In a preferred embodiment the substrate is flat glass or silica.

According to some embodiments, the surface of the substrate is etched using well known techniques to provide for desired surface features. For example, by way of the formation of trenches, v-grooves, mesa structures, or the like, the synthesis regions may be more closely placed within the focus point of impinging light. The surface may also be provided with reflective "mirror" structures for maximization of emission collected therefrom.

Surfaces on the solid substrate will usually, though not always, be composed of the same material as the substrate. Thus, the surface may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed substrate materials. In one embodiment, the surface will be optically transparent and will have surface Si--OH functionalities, such as those found on silica surfaces.

The array of probe sequences may be fabricated on the substrate according to the pioneering techniques disclosed in U.S. Pat. No. 5,143,854 or PCT WO 92/10092, incorporated herein by reference for all purposes. The combination of photolithographic and fabrication techniques may, for example, enable each probe sequence ("feature") to occupy a very small area ("site") on the support. In some embodiments, this feature site may be as small as a few microns or even a single molecule. For example, about 10.sup.5 to 10.sup.6 features may be fabricated in an area of only 12.8 mm.sup.2. Such probe arrays may be of the type known as Very Large Scale Immobilized Polymer Synthesis (VLSIPS.TM.).

Substrate 230 is preferably transparent to a wide spectrum of light. In some embodiments, substrate 230 is made of a conventional microscope glass slide or cover slip. It is preferable that the substrate be as thin as possible while still providing adequate physical support. Preferably, the substrate is less than about 1 mm thick, more preferably less than 0.5 mm thick. Typically the substrate is a microscope glass slide of about 0.7 mm or 700 .mu.m thick. In alternative embodiments, the substrate may be made of quartz or silica.

Substrate 230 is mounted on a flow cell 220. Flow cell 220 is a body having a cavity 221 on a surface thereof. The cavity is between about 50 and 1500 .mu.m deep with a preferred depth of 1000 .mu.m. The bottom of the cavity is preferably light absorbing so as to prevent reflection of impinging light. In addition, the flow cell may be impervious to light.

When mounted to the flow cell, the substrate seals the cavity except for inlet port 223 and outlet port 224. According to a specific embodiment, the substrate is mounted to the flow cell by vacuum pressure generated from a vacuum pump 270. Optionally, one or more gaskets may be placed between the flow cell and substrate and the intervening space is held at vacuum to ensure mating of the substrate to the gaskets.

Reagents, such as fluorescein labeled targets (fluorescence peak at about 530 nm) are injected into the cavity 221 through the inlet port 223 by a pump 240 or by using a syringe. The pump may be, for example, a Masterflex peristaltic pump made by Cole-Parmer Instrument Co or equivalent. Within the cavity, the reagents bind with one or more complementary probes on surface 231 of the substrate. The reagents are circulated into the cavity via inlet port 223 by the pump and exit through the outlet port 224 for recirculation or disposal.

Flow cell 220 permits the substrate to remain in constant contact with reagents during detection, thereby allowing the substrate to be in equilibrium with targets therein. This arrangement also permits the user to manipulate test conditions without dismounting the substrate. In some embodiments, the flow cell provides means for controlling the temperature within the flow cell. The means for controlling temperature may be a recirculating bath device 260 that flows water through channels formed in the flow cell. In the specific embodiment, device 260 is a refrigerated circulating bath with a Rb 232 interface, catalog number 13270-615 distributed by VWR or equivalent. However, means such as a circulating air device, a resistance heater, a peltier device (thermoelectric cooler) or others may also be employed. Computer 190 monitors and controls device 260, thereby maintaining the flow cell at a desired temperature. Computer 190 may be selected from a wide variety of computers including, for example, a Gateway 486DX computer or a similar appropriately programmed computer.

Controlling the temperature in the flow cell is advantageous because temperature affects the chemical reaction between targets and probes. For example, the bond between the targets and probes is generally stronger at lower temperatures. However, if the temperature is too low, the binding affinity between targets and probes may become excessively strong so as to produce apparent (but erroneous) matches. Thus, temperature can be controlled to maximize the binding affinity of complementary targets while minimizing mismatches.

Flow cell 220 is mounted on a x-y-z translation table 250. X represents the horizontal direction; y represents the vertical direction; and z represents the direction into and away from the microscope objective such that focusing may be performed. In some embodiments, the x-y-z translation table may be a Pacific Precision Laboratories Model ST-SL06R-B5M. Movement of the translation table is controlled by computer 190.

A light source 100 generates a beam of light to excite the fluorescein labeled targets in the flow cell. The light source may be a argon laser that generates a beam having a wavelength of about 488 nm, which in some embodiments may be a model 2017 or model 161C manufactured by Spectra-Physics. Other lasers, such as diode lasers, helium neon lasers, dye lasers, titanium sapphire lasers, Nd:YAG lasers or others may also be employed. The laser is directed at surface 231 through an optical train comprised of various optical elements which will be described below in detail.

The beam generated by laser 100 is typically nearly collimated and nearly Gaussian. However, a spatial filter may be optionally located in front of laser 100 to improve the Gaussian profile of the beam. The spatial filter may comprise of a lens 101, a confocal pinhole 103 and a lens 102. Lens 101 and 102, for example, may be 1/2' diameter 50 mm focal length anti-reflection coated plano convex glass lens or equivalent. Both lenses are configured such that both their back focal planes coincide with confocal pinhole 103. Pinhole 103, for example, may have a aperture of 30 .mu.m.

Thereafter, the light passes through a beam splitter 110 to a dichroic mirror 120. The beam splitter may be, for example, a non-polarizing 50% beam splitter cube made by Melles Griot model number 03BSC007 or equivalent while the dichroic mirror may be a LWP-45.degree.S-488R/520T-1025 made by CVI Laser Corp. or equivalent. The functions of the beam splitter cube will later be described in more detail.

In some embodiments, dichroic mirror 120 passes light having a wavelength greater than about 520 nm, but reflects light having a wavelength of about 488 nm. Consequently, the 488 nm light from the laser is reflected by dichroic mirror 120 toward optical lens 130. Optical lens 130, in the specific embodiment, is 1/2' diameter -50 mm focal length anti-reflection coated plano-concave glass lens made by Newport or equivalent. The light then passes through a microscope objective 140 to substrate 230 for magnification of the image sample. Microscope objective 140, in some embodiments, may be a 10.times.0.3NA microscope objective, but other magnifications could also be used. In a preferred embodiment, the distance between lens 130 and microscope objective 140 is about 100 mm.

Microscope objective 140 focuses the light on surface 231, thereby exciting the fluorescein labeled targets. Preferably, the microscope objective produces a spot about 2 .mu.m in diameter in its focal plane. The optical train described in the above embodiments produces a 2 .mu.m diameter focal spot when used with a laser which generates a beam diameter of 1.4 mm, such as the Spectra-Physics model 2017.

In alternative embodiments, the 2 .mu.m spot may be easily obtained when other types of light sources with different beam diameters are used. Since the diameter of the focal spot is inversely proportional to the diameter of the collimated beam produced by lens 102, the desired spot size may be achieved by varying the ratio of the focal lengths of the spatial filter. Alternatively, a beam expander may be used to expand or compress the beam from the light source to obtain the desired spot size. For example, if the laser is a model 161C, which generates a beam diameter of 0.7 mm, a 2 .mu.m diameter focal spot may be achieved if the ratio of the focal lengths of the lenses in the spatial filter is 1:2 instead of 1:1. Thus, by varying the focal lengths of the lenses in the spatial filter and/or using a beam expander, the appropriate excitation spot size may be achieved from various beam diameters.

In a preferred embodiment, the laser power delivered to the sample 50 .mu.W. Depending on the light source used, a variable neutral density filter 310 may be inserted between the laser 100 and the optical train to attenuate the power of the laser to the desired power level.

In response to the excitation light, fluorescein labeled targets in the flow cell fluoresce light having a wavelength greater than about 520 nm. The fluorescence will be collected by the microscope objective 140 and passed to optical lens 130. Optical lens 130 collimates the fluorescence and passes it to dichroic mirror 120. In practice, light collected by microscope objective contains both fluorescence emitted by the fluorescein and 488 nm laser light reflected from the surface 231.

The laser component reflected from the substrate is reflected by dichroic mirror 120 back to beam splitter 110. Beam splitter 110 directs the laser component through a lens 175. The lens, in some embodiments, may be a 1/2' diameter 50 mm focal length anti-reflection coated plano convex glass lens made by Newport, but equivalent thereof may be used. Lens 175 focuses the laser component to a photodiode 170. Preferably, a confocal pinhole 171 is located between lens 175 and photodiode 170. Confocal pinhole transmits substantially only the reflected light originating from the focal plane of the microscope to photodiode 170 while reflected light originating from out-of-focus planes is blocked. In some embodiments confocal pinhole 171 has an aperture of 50 .mu.m. Photodiode 170 generates a voltage corresponding to the intensity of the detected light. Photodiode may be, for example, a 13 DSI007 made by Melles Griot or equivalent, or other light detection devices, such as photomultiplier tube or avalanche photodiode may be used. Output from the detection device is used by computer 190 to focus the laser at a point on surface 231 of substrate 230.

As for the fluorescent component, most of it will pass through the dichroic mirror 120 since its wavelength is greater than about 520 nm. The fluoresced light is then focused by a lens 125 to a photomultiplier tube 160 for detecting the number of photons present therein. Lens 125, in a preferred embodiment, is a 1/2' diameter 50 mm focal length anti-reflection coated plano convex glass lens made by Newport, but equivalent lens may be used. A confocal pinhole 161 may be located adjacent to lens 125. Confocal pinhole transmits florescence originating from the focal plane of the microscope objective and filters out light originating from other planes, such as from the glass or reagent. Accordingly, the signal-to-noise ratio of the fluoresced light is increased. Additionally, a filter 165 is preferably located between photomultiplier tube and confocal pinhole 161. In a specific embodiment, the filter transmits light having a wavelength greater than about 515 nm such as an Omega Optical 515 EFLP. In an alternative embodiment, the filter may transmit light having a wavelength between about 515 and 545 nm such as a 530 DF30 made by Omega Optical. Thus, photomultiplier tube 160 detects substantially only fluoresced light.

In the specific embodiment, photomultiplier tube 160 is a Hamamatsu R4457P photomultiplier tube with Hamamatsu C3866 preamplifier/discriminator. The Photomultiplier tube generates approximately a 2 mV pulse for each photon detected. Each of these 2 mV pulses is converted to a TTL pulse by the preamplifier/discriminator. The TTL pulses, each one corresponding to a photon detected by the photomultiplier tube, are then collected by a data acquisition board 210. The data acquisition board may be a National Instruments "Lab-PC+" or equivalent.

Data acquisition board 210, typically, contains an Intel 8254 or equivalent counter/timer chip. This chip contains three counters, counter 0, counter 1 and counter 2. Counter 0 controls the operations of counters 1 and 2 for collecting data. Preferably, counter 0 is programmed to generate a square wave with a period which is equal to twice the data acquisition time per pixel. The output of counter 0 is coupled to an external circuit board 200 which provides logic for inverting the square wave. In a preferred embodiment, the inverted output of counter 0 is connected to the gate input of counter 2 while the non-inverted output is connected to the gate input of counter 1.

In a preferred embodiment, the data acquisition board is not be able to read or store the fast 10 ns pulses generated by preamplifier/discriminator (it is too fast for the 8254 chip). To solve this problem, external circuit board 200 may additionally provide means for slowing down the pulses. For example, the logic in external circuit board 200 may convert these pulses to 50 ns pulses with at least a 50 ns interval between pulses.

The output of the C3866 preamplifier/discriminator, via external circuit board 200, is connected to the clock inputs of counters 1 and 2. When counter 1 or counter 2 is gated on, it counts pulses generated by the preamplifier/discriminator; when it is gated off, it ceases to count and computer 190 reads the accumulated number of counts therein. After the computer reads the count from either counter 1 or 2, the counter is re-initialized on the first clock pulse after its gate input goes high. The initialization pulse is about a 50 ns pulse that is generated by the logic in the external circuit board 200 about 50 ns after each transition of the square wave signal from counter 0. The data stored in counter 1 or 2 represents the photon count as a function of substrate position.

After data are collected from a region of the substrate, substrate 230 is moved so that light can be directed at a different region on the substrate. The process is repeated until all regions on the substrate have been scanned. Generally, regions that contain a complementary probe will tend to exhibit a higher photon count than regions that do not contain a complementary probe.

Although the above embodiments have been described for use in detecting emissions of fluorescein excited by an 488 nm argon laser, it will be apparent to those skill in art that other dyes and excitation sources may used by simply modifying the elements in the optical train. For example, dichroic mirror 120 may be changed accordingly to pass light having a wavelength comparable to the fluorescence peak of the dye used, but reflect light from the excitation source. Also, filter 165 is changed to pass substantially only light having a wavelength similar to the fluorescence peak of the dye used. In this manner, the detection device can be easily modified to accommodate other types of excitation light and/or dyes.

FIG. 1b illustrates an alternative embodiment of the fluorescence detection device shown in FIG. 1a. FIG. 1b is similar to the one shown in FIG. 1a and the common elements have been numbered with the same reference numerals. The main difference between this embodiment and that of FIG. 1a is that a photodiode 180 is provided to detect a component of the light generated by laser 100. Light generated by the laser, as in FIG. 1a, is directed at the beam splitter. However, a component of this light is directed to photodiode 180. Photodiode 180 generates a voltage value which is proportional to the laser power. This voltage signal is used by the computer 190 to monitor and control the laser power.

FIG. 1c illustrates an alternative embodiment of the fluorescence detection device. FIG. 1c is similar to the embodiment shown in FIG. 1a and the common elements have been numbered with the same reference numerals. However, the embodiment in FIG. 1c provides means for detecting a second fluorescent color. Two color detection is required when two different types of targets, each labeled with a different dye, are exposed to a substrate synthesized with probes. In some embodiments, fluorescein and rhodamine dyes may be used to label two different types of targets respectively. Typically, each dye will have a fluorescence peak at different wavelengths. For example, the fluorescence peak of fluorescein is about 530 nm while that of a typical rhodamine dye is about 580 nm.

To detect the second fluorescent color, a second dichroic mirror 300 is employed. If rhodamine and fluorescein were used, then dichroic mirror 300 is designed to pass light having a wavelength greater than about 570 nm (rhodamine emissions) and reflect light having a wavelength less than about 560 nm (fluorescein emissions). Light with a wavelength less than 560 nm is reflected to a lens 126 and through a confocal pinhole 151. Lens 126, may be equivalent to lens 125 while confocal pinhole 151 may be similar to confocal pinhole 161. Filter 155 transmit the light to a second photomultiplier tube 150. Filter 155 may be an Omega Optical 530DF30 or equivalent that passes light with a wavelength between about 515-545 nm. This ensures that substantially only fluorescein emissions are detected by the photomultiplier 150.

On the other hand, light having a wavelength greater than 570 nm passes through dichroic mirror 300 to a lens 125. Lens 125 then directs the light through pinhole 161 and filter 165 to photomultiplier tube 160. Filter 165 may be a Schott OG570 or equivalent which passes light having a wavelength greater than 570 nm, thereby ensuring substantially only rhodamine emissions are detected by photomultiplier 160.

Output of the preamplifier/discriminator from the photomultiplier tube 150 is processed by the external circuit board 200 before being connected to counters 1b and 2b on the data acquisition board 205. Data collection