Method and apparatus for imaging a sample on a device

• US 6,025,601 A
• Filed: 06/09/1997
• Issued: 02/15/2000
• Est. Priority Date: 09/02/1994
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Walton, Ian, Stern, David, Trulson, Mark, Fodor, Stephen P. A., Rava, Richard, Fiekowsky, Peter
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/871,269
• Time in Patent Office: 981 Days
• Field of Search: 250/585, 250/583, 250/461.2
• US Class Current: 506/37
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6423 Spectral mapping, video display
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

Labeled targets on a support synthesized with polymer sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, can be detected by exposing selected regions of sample 1500 to radiation from a source 1100 and detecting the emission therefrom, and repeating the steps of exposition and detection until the sample is completely examined.

Claims

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 the geometry of said excitation radiation to a line and directing said line at said sample for exciting a plurality of regions thereon, said line causing a labeled material on said sample to emit response radiation, said response radiation having a second wavelength, said first wavelength 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 coupled to said body 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; and

a focuser for automatically focusing said sample in a focal plane of said excitation radiation.

2. The apparatus as recited in claim 1 wherein said body comprises:

a mounting surface;

a cavity in said mounting surface, said first surface mated to said mounting surface for sealing said cavity, said sample being in fluid communication with said cavity, said cavity having a bottom surface comprising a light absorptive material;

an inlet and an outlet being in communication with said cavity such that fluid flowing into said cavity for contacting said sample flows through said inlet and fluid flowing out of said cavity flows through said outlet; and

a temperature controller for controlling the temperature in said cavity.

3. The apparatus as recited in claim 2 wherein said temperature controller comprises a thermoelectric cooler.

4. The apparatus as recited in claim 1 wherein said excitation source is a laser.

5. The apparatus as recited in claim 1 wherein said first wavelength is selected to approximate the absorption maximum of the said labeled material used.

6. The apparatus as recited in claim 1 wherein said excitation optics transform the geometry of said excitation radiation to a line having a length sufficient to excite a strip of said sample with uniform intensity.

7. The apparatus as recited in claim 6 wherein said excitation optics comprises:

a telescope for expanding and collimating said excitation radiation;

a cylindrical telescope for expanding said excitation radiation from said telescope to a desired height; and

a cylindrical lens for focusing said excitation radiation from said cylindrical telescope to a desired width at a focal plane of said cylindrical lens.

8. The apparatus as recited in claim 6 wherein said excitation optics comprises:

a microscope objective for expanding said excitation radiation;

a first lens for collimating said excitation radiation from said microscope objective, said lens comprising an achromatic lens;

a cylindrical telescope for expanding said excitation radiation from said first lens to a desired height; and

a second lens for focusing said excitation radiation from said cylindrical telescope to a desired width at a focal plane of said second lens, said second lens comprising an achromatic lens.

9. The apparatus as recited in claim 1 further comprising a mirror for steering 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.

10. The apparatus as recited in claim 9 wherein said non-zero incident angle is about 45 degrees.

11. The apparatus as recited in claim 1 wherein said collection optics have a magnification power sufficient to achieve a desired image resolution, said collection optics for imaging said response radiation onto said detector, said detector comprising a linear detector array having a length sufficient to detect said response emissions collected by said collection optics.

12. The apparatus as recited in claim 11 wherein said linear detector comprises a CCD linear array.

13. The apparatus as recited in claim 1 wherein said translator comprises an x-y-z translation stage.

14. The apparatus as recited in claim 1 wherein said processor comprises a programmable digital computer.

15. The apparatus as recited in claim 1 further comprising:

a spectral detector for receiving said response emission from said collection optics, said spectral detector detecting a response emission spectrum; and

a filter located in front of said spectral detector, said filter blocking radiation at said first wavelength and passing radiation at other wavelengths.

16. The apparatus as recited in claim 15 wherein said detector comprises a two-dimensional detector array having sufficient size to detect said response emission spectrum from said plurality of regions.

17. The apparatus as recited in claim 16 wherein said two-dimensional detector array comprises a two-dimensional CCD array.

18. The apparatus as recited in claim 1 wherein said excitation source and said excitation optics are configured such that said line travels along a horizontal plane at said support.

23. The method as recited in claim 17 wherein said step of detecting comprises the steps of:

collecting said response radiation through collection optics; and

imaging said response radiation from the collection optics onto radiation detectors, said radiation detectors comprising a linear CCD array.

19. A method for imaging a sample located 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 linear geometry for exciting a plurality of regions 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 a focal plane of said excitation radiation.

20. The method as recited in claim 19 wherein said body comprises a mounting surface having a cavity thereon, said support immobilized on said mounting surface such that said sample is in fluid communication with said reaction chamber, said reaction chamber comprising a inlet and a outlet for flowing fluids into and through said reaction chamber.

21. The method as recited in claim 20 wherein said body further comprises a temperature controller for controlling the temperature in said cavity.

22. The method as recited in claim 19 wherein said step of exciting said sample comprises the step of directing said excitation radiation through excitation optics for transforming the excitation geometry of said excitation radiation to a line, said line having a length sufficient to excite a strip of said sample with uniform energy and a width which is about at least as narrow as the desired image resolution.

24. The method as recited in claim 19 wherein said step of exciting a subsequent plurality of regions comprises the step of translating said sample to allow said excitation radiation to excite a subsequent strip of said sample.

25. The method as recited in claim 19 wherein said step of processing and storing said response radiation comprises the steps of:

a) detecting said response radiation with a detector, said detector generating a signal proportional to amount of radiation it senses;

b) passing said signal to a processor, said processor comprising a digital programmable computer;

c) subtracting a line of dark data stored in said computer from said signal, said line of dark data representing the signal generated by said detector when no radiation is present;

d) storing said data from step c in a memory of said computer;

e) repeating steps a through d until the sample has been completely imaged; and

e) combining the processed data to form a 2-dimensional image of said sample.

26. The method as recited in claim 19 wherein said auto-focusing step comprises the steps of:

a) focusing a first surface of said support;

b) focusing a second surface of said support; and

c) finely focusing said second surface.

27. The method as recited in claim 19 further comprising the step of detecting a response radiation spectrum with a spectrometer, said spectrometer imaging said spectrum onto a two-dimensional CCD array.