Method and apparatus for detection of fluorescently labeled materials

• US 6,141,096 A
• Filed: 03/25/1997
• Issued: 10/31/2000
• Est. Priority Date: 02/10/1994
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stern, David, Fiekowsky, Peter
• Primary Examiner(s): Evans, F. L.
• Application Number:
  US08/823,824
• Time in Patent Office: 1,316 Days
• Field of Search: 356/317, 250/458.1, 250/459.1, 250/461.1, 250/462.1, 250/201.3, 436/518, 435/5
• US Class Current: 356/318
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• 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 2021/174 either absorption-reflection or emission-fluorescence
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Claims

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

a light source;

an optical train for directing light from said light source at said substrate;

a focusing system for focusing said light onto a first surface of said substrate;

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

a flow cell comprising a mounting surface with a cavity therein having said substrate thereon, wherein there is a sealed relationship between said mounting surface and said substrate, whereby said 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.

2. The apparatus as recited in claim 1, wherein said focusing system comprises means for determining a focal plane of the light passing through the optical train and means for providing data for locating said surface of said substrate at said focal plane.

3. The apparatus of claim 1 further comprising:

a photodiode for detecting light reflected from said substrate;

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

a drive connected to said substrate.

4. An apparatus as recited in claim 1, wherein said light source is an excitation light source and said optical train separates reflected excitation light from said surface of the substrate from fluoresced light from said surface.

5. The apparatus of claim 4 wherein the detector 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 1 further comprising a temperature sensor disposed adjacent to or within the cavity of the flow cell and a control system for controlling temperature in said cavity of said flow cell.

7. The apparatus of claim 6 wherein the flow cell defines one or more channels for directing heat exchanging fluid therethrough to control temperature in said cavity.

8. 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 for separating excitation light reflected by said first surface from fluoresced light emitted from said first surface;

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

a second detector for detecting said excitation light reflected from said substrate in response to said excitation light;

a focusing system for focusing said excitation light onto said first surface of said substrate responsive to reflected excitation light detected by the second detector; 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.

9. The apparatus of claim 8 wherein said reflected excitation light and said fluoresced light have first and second wavelengths, respectively, said optical train including an optical device for passing light having the first wavelength and for reflecting light having the second wavelength to separate the reflected excitation light from the fluoresced light.

10. The apparatus of claim 8 further comprising an x-y-z translation system for translating said substrate from a first position to a second position.

11. An apparatus in accordance with claim 8 wherein said first surface of said substrate comprises a nucleic acid probe array.

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

directing an excitation light from an excitation light source at a surface of a substrate;

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

detecting fluoresced light emitted from said substrate in response to said excitation light, said fluoresced light having a second wavelength,

detecting reflected excitation light from said substrate, said detecting fluoresced light and said detecting reflected excitation light comprising separating said reflected excitation light having a first wavelength from said fluoresced light having a second wavelength and separately detecting said light having first and second wavelengths in first and second detectors, respectively;

focusing said excitation light onto a region of said substrate responsive to the intensity of said reflected excitation light detected by said second detector; and

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

13. A method in accordance with claim 12 further comprising a nucleic acid probe affixed to the surface of the substrate.

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

a light source;

an optical train for directing light from said light source at said substrate;

a focusing system for focusing said light onto a first surface of said substrate;

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

a flow cell comprising a mounting surface with a cavity therein having said substrate thereon, wherein there is a sealed relationship between said mounting surface and said substrate, whereby said 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, wherein said substrate comprises a nucleic acid probe array affixed to the first surface of the substrate, the first surface being in fluid communication with said cavity.

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

a light source;

an optical train for directing light from said light source at said substrate;

a focusing system for focusing said light onto said surface;

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

a flow cell comprising a mounting surface with a cavity therein having said substrate mounted thereon, wherein there is a sealed relationship between said mounting surface and said substrate, whereby said surface of said substrate is in fluid communication with said cavity.