Systems and methods for detection of labeled materials

• US 6,597,000 B2
• Filed: 07/08/2002
• Issued: 07/22/2003
• Est. Priority Date: 05/16/1996
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stern, David
• Primary Examiner(s): GAGLIARDI, ALBERT J
• Application Number:
  US10/191,420
• Publication Number:
  US 20020185610A1
• Time in Patent Office: 379 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.2, 250/201.3, 356/344, 356/317
• US Class Current: 506/12
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0072 details concerning resolution or correction, including general design of CSOM objectives
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/0084 time-scale detection, e.g. strobed, ultra-fast, heterodyne detection

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 marked regions of sample to radiation from a source and detecting the emission therefrom, and repeating the steps of exposition and detection until the sample is completely examined.

Claims

1. A system for detecting marked regions on a surface, comprising:

an excitation radiation source;

a focusing optics constructed and arranged to focus radiation from said excitation radiation source onto a surface region of a wafer including several chips with probe sequences;

a focusing system constructed and arranged to displace said wafer to bring at least a portion of said wafer in focus with respect to said focused radiation;

a radiation direction system constructed and arranged to scan said excitation radiation across several surface regions;

a translation stage upon which said wafer is mounted, said translation stage being moveable in at least one dimension and being arranged with said radiation direction system to scan a plurality of different chips on said wafer;

a detector for detecting fluorescent radiation emitted from said surface region in response to said excitation radiation; and

a data acquisition system for recording an amount of said fluorescent radiation detected as a function of a position on said surface region from which said fluorescent radiation was emitted.

2. The system of claim 1, wherein said focusing optics includes an objective lens.

3. The system of claim 2, wherein said objective lens has a selected ratio of a scanning field diameter to a focused spot diameter.

4. The system of claim 1, wherein said translation stage is moveable in said at least one dimension that is perpendicular to an optical axis of said objective lens.

5. The system of claim 4 wherein said focusing system includes an autofocus system comprising second focusing optics constructed to focus said excitation radiation reflected from said surface region through a confocal pinhole onto a photodetector and cooperatively arranged with said translation stage to move said surface region into a focal plane of said first-mentioned focusing optics.

6. The system of claim 1, wherein said translation stage is moveable in a dimension parallel to an optical axis of said objective lens and in a dimension perpendicular to said optical axis of said objective lens.

7. The system of claim 1 wherein said focusing system includes an autofocus system constructed and arranged to place said surface region in a focal plane of said focusing optics.

8. The system of claim 7, wherein said autofocus system is constructed and arranged to move said surface region into said focal plane by detecting a maximum value of reflected excitation radiation reaching said photodetector while moving said translation stage in said dimension parallel to said optical axis of said objective lens.

9. The system of claim 1, wherein said objective lens has a scanning field diameter is greater than about 10 mm.

10. The system of claim 1, wherein said objective lens has a numerical aperture greater than about 0.25.

11. The system of claim 1, wherein said focusing optics has said ratio of scanning field diameter to focused spot diameter of greater than 3000.

12. The system of claim 1, wherein said focusing optics focuses said excitation radiation in a spot having a diameter of less than about 10 μ

m.

13. The system of claim 1, wherein said focusing optics focuses said excitation radiation in a spot having a diameter of less than about 5 μ

m.

14. The system of claim 1, wherein said focusing optics focuses said excitation radiation in a spot having a diameter of less than about 3 μ

m.

15. The system of claim 1, wherein said focusing optics is achromatic.

16. The system of claim 1, wherein said radiation direction system is capable of scanning a spot across the substrate at a rate of at least 10 image lines/second.

17. The system of claim 1, wherein said radiation direction system is capable of scanning a spot across the substrate at a rate of at least 30 image lines/second.

18. The system of claim 1 including collection optics for collecting fluorescent radiation emitted from said surface region and said excitation radiation reflected from said surface region, and including separation optics for separating said fluorescent radiation from said excitation radiation and focusing said fluorescent radiation through a confocal pinhole to said detector.

19. A method of scanning a polymer array having a plurality of different polymer sequences, each of said different polymer sequences being immobilized on a chip in a different known location, to identify which polymer sequence on said array is bound by a target molecule, the method comprising:

focusing excitation radiation from an excitation radiation source onto a surface region of a wafer including several chips with polymer sequences;

displacing said wafer to bring at least a portion of said wafer in focus with respect to said focused radiation;

scanning said focused excitation radiation across several said surface regions;

translating said wafer in at least one dimension by a translation stage upon which said wafer is mounted, said scanning and translating enabling a scan of a plurality of different chips on said wafer;

detecting fluorescent radiation from said surface region in response to said excitation radiation; and

recording an amount of said fluorescent radiation detected as a function of a position on said surface region from which said fluorescent radiation was emitted.

20. The method of claim 19, wherein said focusing includes using an objective lens.

21. The method of claim 20, wherein said objective lens has a selected ratio of a scanning field diameter to a focused spot diameter.

22. The method of claim 20, wherein said translating includes moving in said at least one dimension that is perpendicular to an optical axis of said objective lens.

23. The method of claim 20, wherein said translating includes moving in a dimension parallel to an optical axis of said objective lens and moving in a dimension perpendicular to said optical axis of said objective lens.

24. The method of claim 19 wherein said displacing said wafer includes autofocusing by placing said surface region in a focal plane of said focusing optics.

25. The method of claim 19 wherein said displacing said wafer includes autofocusing by placing said surface region in a focal plane of said focusing optics by translating said wafer.

26. The method of claim 19, wherein said autofocusing includes moving said surface region and detecting a maximum value of reflected excitation radiation reaching a photodetector while performing said moving in said dimension parallel to said optical axis of said objective lens.

27. The method of claim 19 including collecting fluorescent radiation emitted from said surface region, collecting said excitation radiation reflected from said surface region, and separating said fluorescent radiation from said excitation radiation.

28. The method of claim 27 including focusing said fluorescent radiation through a confocal pinhole to a detector detecting said fluorescent radiation.

29. The method of claim 19, wherein said polymer sequence comprises a plurality of different oligonucleotide sequences immobilized on said surface region of said chip in different known locations.

30. The method of claim 19 further comprising focusing the excitation radiation source to a spot having a diameter of about 3 μ

m.