Method and apparatus for imaging a sample on a device

What is claimed is:

1. An apparatus for imaging a sample located on a support, said apparatus comprising:

a body for immobilizing said support, said support comprising at least a first surface having said sample thereon;

an electromagnetic radiation source for generating excitation radiation having a first wavelength;

excitation optics for transforming a geometry of said excitation radiation to an excitation line, said excitation line having a focal plane, and directing said excitation line at said sample for exciting a plurality of regions thereon, said excitation line causing a labeled material on said sample to emit a response radiation, said response radiation having a second wavelength, said first wavelength being different from said second wavelength;

collection optics for collecting said response radiation from said plurality of regions;

a detector for sensing said response radiation received by said collection optics, said detector generating a signal proportional to the amount of radiation sensed thereon, said signal representing an image associated with said plurality of regions from said sample;

a translator for allowing a subsequent plurality of regions on said sample to be excited;

a processor for processing and storing said signal so as to generate a 2-dimensional image of said sample;

a mirror for directing said excitation radiation to excite said plurality of regions at a non-zero incident angle such that said response radiation and said excitation line reflected from said support are decoupled from each other; and

a focuser for automatically focusing said sample in said focal plane of said excitation line, said focuser comprising first focusing optics for receiving said reflected excitation line and focusing said reflected excitation line to a first spot, a first slit located such that said first spot traverses said first slit when said translator moves said support in a direction relative to said excitation line, said first spot located at substantially a center of said first slit when said support is substantially in said focal plane, and a first radiation detector located behind said first slit for generating a signal proportional to an amount of radiation detected, said amount of radiation being greatest when said support is located in said focal plane.

2. The apparatus as recited in claim 1, wherein said focusing optics comprise:

a cylindrical lens for collimating said line reflected from said support; and

a lens for focusing said line collimated by said cylindrical lens to a spot.

3. The apparatus as recited in claim 1, further comprising a position adjustor for locating said support automatically in a substantially perpendicular position relative to an optical axis of said collection optics, said position adjustor comprising:

a tilt stage for rotating said body until it reaches said substantially perpendicular position relative to the optical axis of said collection optics;

a beam splitter for directing a portion of said reflected excitation line from said first focusing optics;

second focusing optics for receiving said portion of said reflected excitation line from said beam splitter and focusing said reflected excitation line to a second spot;

a second slit located such that said second spot traverses said slit perpendicularly when said tilt stage rotates said support, said second spot located at substantially a center of said second slit when said support is substantially perpendicular relative to the optical axis of said collection optics; and

a second radiation detector located behind said second slit for generating a signal proportional to an amount of radiation detected, said amount of radiation being greatest when said support is located substantially perpendicular relative to the optical axis of said collection optics.

4. The apparatus as recited in claim 3, wherein said substantially perpendicular position is substantially vertical.

5. The apparatus as recited in claim 3, wherein said tilt stage is controlled by said processor.

6. The apparatus as recited in claim 3, wherein said position adjustor comprises an air bearing system, said air bearing system comprising:

an optics head comprising a substantially planar plate, said planar plate comprising a first surface having a plurality of holes disposed therein, said plurality of holes being in communication with an air inlet;

a pump connected to said air inlet for flowing air through said plurality of holes;

a valve for regulating a flow of air from said air pump through said plurality of holes; and

an air ballast to dampen air pressure variations, said optics head maintained in a relative position by air pressure through said plurality of holes such that said support is maintained in a substantially perpendicular position relative to said optical axis of said collection optics.

7. The apparatus as recited in claim 6, wherein said electromagnetic radiation source, excitation optics, collection optics and sensing detector are enclosed in said optics head.

8. The apparatus of claim 1, further comprising a support immobilized on said body, said support comprising at least a first surface having a sample thereon, said sample on said first surface of said support comprising a plurality of different oligonucleotide sequences, each of said plurality of different oligonucleotide sequences being in a separate known location on said first surface of said support.

9. The apparatus of claim 8, wherein said sample comprises more than 100 different oligonucleotide sequences in a separate known location on said first surface of said support.

10. The apparatus of claim 8, wherein said sample comprises more than 1000 different oligonucleotide sequences in a separate known location on said first surface of said support.

11. The apparatus of claim 8, wherein said sample comprises more than 10,000 different oligonucleotide sequences in a separate known location on said first surface of said support.

12. The apparatus of claim 8, wherein said sample comprises more than 100,000 different oligonucleotide sequences in a separate known location on said first surface of said support.

13. The apparatus of claim 8, wherein said support is glass.

14. A method of imaging a sample on a support, said method comprising the steps of:

immobilizing said support on a body;

exciting said sample on said support with an excitation radiation having a first wavelength from an electromagnetic radiation source, said excitation radiation having a focal plane and a linear geometry for exciting a plurality of region on said sample;

detecting a response radiation having a second wavelength in response to said excitation radiation, said response radiation representing an image of said plurality of regions;

exciting a subsequent plurality of regions on said sample;

processing and storing said response radiation to generate a 2-dimensional image of said sample; and

auto-focusing said sample in said focal plane of said excitation radiation, said step of autofocusing comprising the steps of focusing a first surface of said support, coarse focusing on a second surface said support and finely focusing said second surface, wherein said step of focusing said first surface comprises the steps of directing said excitation radiation at said first surface of said support, said excitation radiation being reflected by said support, focusing said reflected excitation radiation through a slit, detecting an amount of reflected excitation radiation passing through said slit, said slit configured such that said reflected excitation radiation is located substantially at a center of said slit when said first surface is located in substantially the focal plane of said excitation radiation, determining if said amount of reflected excitation radiation passing through said slit has peaked, moving said support closer relative to said electromagnetic radiation source and repeating the directing, focusing, detecting, determining, and moving steps until said amount of reflected excitation radiation passing through said slit has peaked.

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

first moving said support closer relative to said electromagnetic radiation source, the distance which the said support is moved being equal to about half the thickness of said support;

directing said excitation radiation at said support, said excitation radiation being reflected by said support;

focusing said excitation radiation reflected by said support through said slit;

detecting an amount of said excitation radiation reflected by said support and passing through said slit;

determining if said amount of excitation radiation reflected by said support and passing through said slit has peaked;

second moving said support closer relative to said electromagnetic radiation source and repeating the directing, focusing, detecting, determining and moving steps until said amount of excitation radiation reflected by said support and passing through said slit has peaked.

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

directing said excitation radiation at said support, said excitation radiation being reflected by said support;

focusing said excitation radiation reflected by said support through said slit;

detecting said amount of excitation radiation reflected by said support and passing through said slit, said slit configured such that said excitation radiation reflected by said support is located substantially at a center of said slit when said second surface is located substantially in the focal plane of said excitation radiation;

determining if said amount of excitation radiation reflected by said support and passing through said slit has peaked; and

moving said support farther relative to said electromagnetic radiation source and repeating the directing, focusing, detecting, determining and moving steps until said amount of excitation radiation reflected by said support passing through said slit has reached a desired value.

17. The method of claim 14, wherein said sample excited in said exciting step comprises a plurality of different oligonucleotide sequences, each of said plurality of different oligonucleotide sequences being in a separate known location on said second surface of said support.

18. The method of claim 17, wherein said sample comprises more than 100 different oligonucleotide sequences in a separate known location on said second surface of said support.

19. The method of claim 17, wherein said sample comprises more than 1000 different oligonucleotide sequences in a separate known location on said second surface of said support.

20. The method of claim 17, wherein said sample comprises more than 10,000 different oligonucleotide sequences in a separate known location on said second surface of said support.

21. The method of claim 17, wherein said sample comprises more than 100,000 different oligonucleotide sequences in a separate known location on said second surface of said support.

22. The method of claim 17, wherein said support is glass.

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COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION

The present invention relates to the field of imaging. In particular, the present invention provides methods and apparatus for high speed imaging of a sample containing labeled markers with high sensitivity and resolution.

Methods and systems for imaging samples containing labeled markers such as confocal microscopes are commercially available. These systems, although capable of achieving high resolution with good depth discrimination, have a relatively small field of view. In fact, the system's field of view is inversely related to its resolution. For example, a typical 40.times. microscope objective, which has a 0.25 .mu.m resolution, has a field size of only about 500 .mu.m. Thus, confocal microscopes are inadequate for applications requiring high resolution and large field of view simultaneously.

Other systems, such as those discussed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO 92/10092, and U. S. patent application Ser. No. 08/195,889, filed Feb. 10, 1994, incorporated herein by reference for all purposes, are also known. These systems include an optical train which directs a monochromatic or polychromatic light source to about a 5 micron (.mu.m) diameter spot at its focal plane. A photon counter detects the emission from the device in response to the light. The data collected by the photon counter represents one pixel or data point of the image. Thereafter, the light scans another pixel as the translation stage moves the device to a subsequent position.

As disclosed, these systems resolve the problem encountered by confocal microscopes. Specifically, high resolution and a large field of view are simultaneously obtained by using the appropriate objective lens and scanning the sample one pixel at a time. However, this is achieved by sacrificing system throughput. As an example, an array of material formed using the pioneering fabrication techniques, such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), U.S. patent application Ser. No. 08/143,312, and U.S. patent application Ser. No. 08/255,682, incorporated herein by reference for all purposes, may have about 10.sup.5 sequences in an area of about 13 mm.times.13 mm. Assuming that 16 pixels are required for each member of the array (1.6.times.10.sup.6 total pixels), the image can take over an hour to acquire.

In some applications, a full spectrally resolved image of the sample may be desirable. The ability to retain the spectral information permits the use of multi-labeling schemes, thereby enhancing the level of information obtained. For example, the microenvironment of the sample may be examined using special labels whose spectral properties are sensitive to some physical property of interest. In this manner, pH, dielectric constant, physical orientation, and translational and/or rotational mobility may be determined.

From the above, it is apparent that improved methods and systems for imaging a sample are desired.

SUMMARY OF THE INVENTION

Methods and systems for detecting a labeled marker on a sample located on a support are disclosed. The imaging system comprises a body for immobilizing the support. Excitation radiation, from an excitation source having a first wavelength, passes through excitation optics. The excitation optics cause the excitation radiation to excite a region on the sample. In response, labeled material on the sample emits radiation which has a wavelength that is different from the excitation wavelength. Collection optics then collect the emission from the sample and image it onto a detector. The detector generates a signal proportional to the amount of radiation sensed thereon. The signal represents an image associated with the plurality of regions from which the emission originated. A translator is employed to allow a subsequent plurality of regions on said sample to be excited. A processor processes and stores the signal so as to generate a 2-dimensional image of said sample.

In one embodiment, excitation optics focus excitation light to a line at a sample, simultaneously scanning or imaging a strip of the sample. Surface bound labeled targets from the sample fluoresce in response to the light. Collection optics image the emission onto a linear array of light detectors. By employing confocal techniques, substantially only emission from the light's focal plane is imaged. Once a strip has been scanned, the data representing the 1-dimensional image are stored in the memory of a computer. According to one embodiment, a multi-axis translation stage moves the device at a constant velocity to continuously integrate and process data. As a result, a 2-dimensional image of the sample is obtained.

In another embodiment, collection optics direct the emission to a spectrograph which images an emission spectrum onto a 2-dimensional array of light detectors. By using a spectrograph, a full spectrally resolved image of the sample is obtained.

The systems may include auto-focusing feature to maintain the sample in the focal plane of the excitation light throughout the scanning process. Further, a temperature controller may be employed to maintain the sample at a specific temperature while it is being scanned. The multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection are managed by an appropriately programmed digital computer.

In connection with another aspect of the invention, methods for analyzing a full spectrally resolved image are disclosed. In particular, the methods include, for example, a procedure for deconvoluting the spectral overlap among the various types of labels detected. Thus, a set of images, each representing the surface densities of a particular label can be generated.

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. 1 shows a block diagram of an imaging system;

FIG. 2 illustrates how the imaging system achieves good depth discrimination;

FIG. 3 shows the imaging system according to the present invention;

FIGS. 4a-4d show a flow cell on which a substrate is mounted;

FIG. 5 shows a agitation system;

FIG. 6 is a flow chart illustrating the general operation of the imaging system;

FIGS. 7a-7b are flow charts illustrating the steps for focusing the light at the sample;

FIG. 8 is a flow chart illustrating in greater detail the steps for acquiring data;

FIG. 9 shows an alternative embodiment of the imaging system;

FIG. 10 shows the axial response of the imaging system of FIG. 9;

FIGS. 11a-11b are flow charts illustrating the general operations of the imaging system according to FIG. 9;

FIGS. 12a-12b are flow charts illustrating the steps for plotting the emission spectra of the acquired image;

FIG. 12c shows the data structure of the data file according to the imaging system in FIG. 9;

FIG. 13 shows the normalized emission spectra of four fluorophores whose emission spectra are shown in FIG. 17, and scaled according to the steps set forth in FIG. 12 a after they have been normalized;

FIG. 14 is a flow chart illustrating the steps for image deconvolution;

FIG. 15 shows the layout of the probe sample;

FIG. 16 shows examples of monochromatic images obtained by the imaging system of FIG. 9;

FIGS. 17-18 show the emission spectra obtained by the imaging system of FIG. 9;

FIG. 19 shows the emission cross section matrix elements obtained from the emission spectra of FIG. 13;

FIG. 20 shows examples of images representing the surface density of the fluorophores; and

FIG. 21 shows an alternative embodiment of an imaging system.

DESCRIPTION OF THE PREFERRED EMBODIMENT CONTENTS

I. Definitions

II. General

a. Introduction

b. Overview of the Imaging System

III. Detailed Description of One Embodiment of the Imaging System

a. Detection Device

b. Data acquisition

IV. Detailed Description of an Alternative Embodiment of the Imaging System

a. Detection Device

b. Data Acquisition

c. Postprocessing of the Monochromatic Image Set

d. Example of spectral deconvolution of a 4-fluorophore system

V. Detailed Description of Another Embodiment of the Imaging System

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 surface-immobilized 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., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

3. Target: 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, oligonucleotides, 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. General

a. Introduction

The present invention provides methods and apparatus for obtaining a highly sensitive and resolved image at a high speed. The invention will have a wide range of uses, particularly, those requiring quantitative study of a microscopic region from within a larger region, such as 1 .mu.m.sup.2 over 100 mm.sup.2. For example, the invention will find application in the field of histology (for studying histochemical stained and immunological fluorescent stained images), video microscopy, or fluorescence in situ hybridization. In one application, the invention herein is used to image an array of probe sequences fabricated on a support.

The support 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 skill 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-0H functionalities, such as those found on silica surfaces.

The array of probe sequences may be fabricated on the support according to the pioneering techniques disclosed in U.S. Pat. No. 5,143,854, PCT WO 92/10092, or U. S. application Ser. No. 07/624,120, filed Dec. 6, 1990, 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.).

The probe arrays will have a wide range of applications. For example, the probe arrays may be designed specifically to detect genetic diseases, either from acquired or inherited mutations in an individual DNA. These include genetic diseases such as cystic fibrosis, diabetes, and muscular dystrophy, as well as acquired diseases such as cancer (P53 gene relevant to some cancers), as disclosed in U.S. Pat. application Ser. No. 08/143,312, already incorporated by reference.

Genetic mutations may be detected by a method known as sequencing by hybridization. In sequencing by hybridization, a solution containing one or more targets to be sequenced (i.e., samples from patients) contacts the probe array. The targets will bind or hybridize with complementary probe sequences. Generally, the targets are labeled with a fluorescent marker, radioactive isotopes, enzymes, or other types of markers. Accordingly, locations at which targets hybridize with complimentary probes can be identified by locating the markers. Based on the locations where hybridization occur, information regarding the target sequences can be extracted. The existence of a mutation may be determined by comparing the target sequence with the wild type.

The interaction between targets and probes can be characterized in terms of kinetics and thermodynamics. As such, it may be necessary to interrogate the array while in contact with a solution of labeled targets. Consequently, the detection system must be extremely selective, with the capacity to discriminate between surface-bound and solution-born targets. Also, in order to perform a quantitative analysis, the high-density volume of the probe sequences requires the system to have the capacity to distinguish between each feature site.

b. Overview of the Imaging System

An image is obtained by detecting the electro-magnetic radiation emitted by the labels on the sample when it is illuminated. Emission from surface-bound and solution-free targets is distinguished through the employment of confocal and auto-focusing techniques, enabling the system to image substantially only emission originating from the surface of the sample. Generally, the excitation radiation and response emission have different wavelengths. Filters having high transmissibility in the label's emission band and low transmissibility in the excitation wavelength may be utilized to virtually eliminate the detection of undesirable emission. These generally include emission from out-of-focus planes or scattered excitation illumination as potential sources of background noise.

FIG. 1 is an optical and electronic block diagram illustrating the imaging system according to the present invention. Illumination of a sample 1500 may be achieved by exposing the sample to electromagnetic radiation from an excitation source 1100. Various excitation sources may be used, including those which are well known in the art such as an argon laser, diode laser, helium-neon laser, dye laser, titanium sapphire laser, Nd:YAG laser, arc lamp, light emitting diodes, any incandescent light source, or other illuminating device.

Typically, the source illuminates the sample with an excitation wavelength that is within the visible spectrum, but other wavelengths (i.e., near ultraviolet or near infrared spectrum) may be used depending on the application (i.e., type of markers and/or sample). In some embodiments, the sample is excited with electromagnetic radiation having a wavelength at or near the absorption maximum of the species of label used. Exciting the label at such a wavelength produces the maximum number of photons emitted. For example, if fluorescein (absorption maximum of 488 nm) is used as a label, an excitation radiation having a wavelength of about 488 nm would induce the strongest emission from the labels.

In instances where a multi-labeling scheme is utilized, a wavelength which approximates the mean of the various candidate labels' absorption maxima may be used. Alternatively, multiple excitations may be performed, each using a wavelength corresponding to the absorption maximum of a specific label. Table I lists examples of various types of fluorophores and their corresponding absorption maxima.

TABLE I ______________________________________ Candidate Fluorophores Absorption Maxima ______________________________________ Fluorescein 488 nm Dichloro-fluorescein 525 nm Hexachloro-fluorescein 529 nm Tetramethylrhodamine 550 nm Rodamine X 575 nm Cy3 .TM. 550 nm Cy5 .TM. 650 nm Cy7 .TM. 750 nm IRD40 785 nm ______________________________________

The excitation source directs the light through excitation optics 1200, which focus the light at the sample. The excitation optics transform the light into a "line" sufficient to illuminate a row of the sample. Although the Figure illustrates a system that images one vertical row of the sample at a time, it can easily be configured to image the sample horizontally or to employ other detection scheme. In this manner, a row of the sample (i.e., multiple pixels) may be imaged simultaneously, increasing the throughput of the imaging systems dramatically.

Generally, the excitation source generates a beam with a Gaussian profile. In other words, the excitation energy of the line peaks flatly near the center and diminishes therefrom (i.e., non-uniform energy profile). Illuminating the sample with a non-uniform energy profile will produce undesirable results. For example, the edge of the sample that is illuminated by less energetic radiation would appear more dim relative to the center. This problem is resolved by expanding the line to permit the central portion of the Gaussian profile to illuminate the sample.

The width of the line (or the slit aperture) determines the spatial resolution of the image. The narrower the line, the more resolved the image. Typically, the line width is dictated by the feature size of sample. For example, if each probe sequence occupies a region of about 50 .mu.m, then the minimum width is about 50 .mu.m. Preferably, the width should be several times less than the feature size to allow for oversampling.

Excitation optics may comprise various optical elements to achieved the desired excitation geometry, including but not limited to microscope objectives, optical telescopes, cylindrical lens, cylindrical telescopes, line generator lenses, anamorphic prisms, combination of lenses, and/or optical masks. The excitation optics may be configured to illuminate the sample at an angle so as to decouple the excitation and collection paths. As a result, the burden of separating the light paths from each other with expensive dichroic mirrors or other filters is essentially eliminated. In one embodiment, the excitation radiation illuminates the sample at an incidence of about 45.degree.. This configuration substantially improves the system's depth discrimination since emission from out-of-focus planes is virtually undetected. This point will subsequently be discussed in more detail in connection with FIG. 2.

As the incident light is reflected from the sample, it passes through focusing optics 1400, which focus the reflected illumination line to a point. A vertical spatial slit 1405 and light detector 1410 are located behind the focusing optics. Various light detectors may be used, including photodiodes, avalanche photodiodes, phototransistors, vacuum photodiodes, photomultiplier tubes, and other light detectors. The focusing optics, spatial slit, and light detector serve to focus the sample in the focal plane of the excitation light. In one embodiment, the light is focused at about the center of the slit when the sample is located in the focal plane of the incident light. Using the light detector to sense the energy, the system can determine when the sample is in focus. In some applications, the slit may be eliminated by employing a split photodiode (bi-cell or quadrant detector), position-sensitive photodiode, or position-sensitive photomultiplier.

The line illumination technique presents certain concerns such as maintaining the plane of the sample perpendicular to the optical axis of the collection optics. If the sample is not aligned properly, image distortion and intensity variation may occur. Various methods, including shims, tilt stage, gimbal mount, goniometer, air pressure or pneumatic bearings or other technique may be employed to maintain the sample in the correct orientation. In one embodiment, a beam splitter 1420 may be strategically located to direct a portion of the beam reflected from the sample. A horizontal spatial slit 1425 and light detector 1430, similar to those employed in the auto-focusing technique, may be used to sense when the plane of the sample is perpendicular to the optical axis of the collection optics.

In response to the excitation light, the labeled targets fluoresce (i.e.,