Method and apparatus for imaging a sample on a device

• US 5,578,832 A
• Filed: 09/02/1994
• Issued: 11/26/1996
• 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): Fields, Carolyn E.
• Assistant Examiner(s): Glick, Edward J.
• Application Number:
  US08/301,051
• Time in Patent Office: 816 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.1, 250/462.1, 250/201.2, 250/201.3, 250/201.4
• US Class Current: 506/39
• 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

The present invention provides methods and systems for detecting a labeled marker on a sample located on a support. The imaging system comprises a body for immobilizing the support, an excitation radiation source and excitation optics to generate and direct the excitation radiation at the sample. In response, labeled material on the sample emits radiation which has a wavelength that is different from the excitation wavelength, which radiation is collected by collection optics and imaged onto a detector which generates an image of the sample.

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 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.