System and methods for detection of labeled materials

• US 6,207,960 B1
• Filed: 04/21/1999
• Issued: 03/27/2001
• 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): Hannaher, Constantine
• Assistant Examiner(s): GAGLIARDI, ALBERT J
• Application Number:
  US09/295,214
• Time in Patent Office: 706 Days
• Field of Search: 250/458.1, 250/459.1, 250/206.3, 250/461.2, 356/344, 356/317
• US Class Current: 506/39
• 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 of a substrate, the system comprising:

an excitation radiation source;

a focusing system for focusing radiation from said excitation radiation source onto a selected region of said surface of said substrate, said focusing system including an objective lens having a ratio of scanning field diameter to focused spot diameter of greater than about 2000, and a numerical aperture greater than about 0.2;

a reciprocating radiation direction system comprising a mirror selected from the group consisting of a galvanometric mirror, angularly oscillating mirror or a rotating polyhedral mirror for scanning said focused excitation radiation across said surface of said substrate at a rate of at least 20 image lines/second;

a detector for detecting an emission from said surface of said substrate in response to said excitation radiation, wherein said objective lens receives said emission and transmits the emission to the detector; and

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

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

3. The system of claim 1, wherein said focusing system has a ratio of scanning field diameter to focused spot diameter of greater than 4000.

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

m.

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

m.

6. The system of claim 1, wherein said focusing system focuses said excitation radiation on said surface of said substrate in a spot having a diameter of about 3 μ

m.

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

8. The system of claim 1, wherein said scanning field diameter is about 14 mm.

9. The system of claim 1, wherein said numerical aperture is greater than about 0.25.

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

11. 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.

12. 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.

13. The system of claim 1, further comprising a translation stage upon which said substrate is mounted, said translation stage being moveable in at least one dimension perpendicular to an optical axis of said objective lens.

14. The system of claim 1, further comprising an autofocus system for placing said surface of said substrate in a focal plane of said focusing system.

15. The system of claim 1, further comprising a translation stage upon which said substrate is mounted, said translation stage being moveable in at least one dimension parallel to an optical axis of said focusing system and at least one dimension perpendicular to said optical axis of said focusing system.

16. The system of claim 1 further comprising a collection system comprising:

collection optics for collecting fluorescent radiation emitted from said surface of said substrate and for collecting said excitation radiation reflected from said surface of said substrate;

separation optics for separating said fluorescent radiation from said excitation radiation and focusing said fluorescent radiation through a confocal pinhole; and

a recorder responsive to said fluorescent radiation, for recording an amount of said fluorescence focused through said confocal pinhole.

17. The system of claim 1, wherein said surface of said substrate comprises a plurality of distinct polymer sequences in different known locations on said surface.

18. The system of claim 17, wherein each of said different polymer sequences is contained in a feature having at least one of a width or length dimension of less than about 50 μ

m.

19. The system of claim 1 further comprising a processor for processing and storing a signal so as to generate a 2-dimensional image of said substrate.

20. The system of claim 1 further comprising a body for immobilizing said substrate, the substrate having at least a first surface with a sample thereon, the body comprising:

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.

21. The system of claim 20 wherein said temperature controller comprises a thermoelectric cooler.

22. A system for detecting marked regions on a surface of a substrate, the system comprising:

an excitation radiation source;

first focusing optics for focusing said excitation radiation on said surface of said substrate in a spot having a diameter no greater than 5 μ

m, said focusing optics including an objective lens having a ratio of scanning field diameter to focussed spot diameter of greater than about 2000;

a reciprocating radiation direction system comprising a mirror selected from the group consisting of a galvanometric mirror, angularly oscillating mirror or rotational polyhedral mirror for scanning said spot linearly across said surface of said substrate at a rate of at least 20 image lines/second said spot having a travel distance of at least 10 mm;

an optical train for separating emissions from said surface of said substrate from excitation radiation reflected from said surface of said substrate, said optical train including said objective lens, said objective lens receiving both said reflected excitation radiation and said emissions therethrough; and

an autofocus system for automatically placing said surface of said substrate in a focal plane of said first focusing optics.

23. The system of claim 22, wherein said optical train includes a dichroic beam splitter for separating fluorescence emitted from said surface of said substrate from excitation radiation reflected from said surface of said substrate.

24. The system of claim 22, wherein said autofocus system comprises:

second focusing optics for focusing said excitation radiation reflected from said surface of said substrate through a confocal pinhole onto a photodiode; and

a translation stage upon which a substrate is mounted, said stage being capable of moving in a direction parallel with an optical axis of said first focusing optics for moving said substrate into a focal plane of said first focusing optics.

25. The system of claim 22 further comprising

a detector for detecting an emission from said surface of said substrate in response to said excitation radiation, and a data acquisition system for recording an amount of said emission detected as a function of a position on said surface of said substrate from which said emission was emitted.

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

focusing an excitation radiation source upon said surface of said substrate with an objective lens having a ratio of scanning field diameter to focussed spot diameter of greater than about 2000;

scanning said excitation radiation across said surface of said substrate at a speed of at least about 20 image lines/second said scanning comprising using a mirror selected from the group consisting of a galvanometric mirrors, angularly oscillating mirror or rotating polyhedral mirror to direct said excitation radiation through said objective lens and across said substrate;

collecting emissions from said surface of said substrate in response to said excitation radiation with said objective lens;

collecting excitation radiation reflected from said substrate, thereby focussing said emissions; and

recording said emissions as a function of a position on said surface of said substrate, said position indicating the polymer sequence on said array is bound by a target molecule.

27. The method of claim 26, wherein said polymer array comprises a plurality of different oligonucleotide sequences immobilized on said surface of said substrate in different known locations.

28. The method of claim 26 further comprising focusing the excitation radiation source to a spot having a diameter of about 3 μ

m and scanning the spot in linear direction across the substrate for at least 14 mm.

29. The method of claim 26 further comprising the steps of:

immobilizing said substrate on a body;

exciting said surface on said substrate with an excitation radiation having a first wavelength from an electromagnetic radiation source, said excitation radiation being focussed on said substrate by an objective lens having a ratio of scanning diameter to focussed spot diameter of greater than about 2000;

exciting performed at a rate of at least 20 image lines/second;

detecting response radiation having a second wavelength, said response radiation being generated in response to said excitation radiation, said response radiation representing an image of a first plurality of regions, said response radiation being focussed through said objective lens;

exciting a subsequent plurality of regions on said sample;

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

separating excitation radiation reflected from said substrate from said response radiation; and

detecting said reflected excitation radiation, thereby auto-focusing said sample in a focal plane of said excitation radiation.

30. The method as recited in claim 29 wherein said body comprises a mounting surface having a cavity thereon, said substrate being immobilized on said mounting surface such that said surface is in fluid communication with said reaction chamber, a reaction chamber comprising a inlet and a outlet for flowing fluids into and through said reaction chamber.

31. The method as recited in claim 30 wherein said body further comprises a temperature controller for controlling the temperature in said cavity.

32. The method as recited in claim 26 wherein said excitation radiation is scanned across said surface of said substrate at a speed of at least about 10 image lines/second.

33. The method as recited in claim 26 wherein said excitation radiation is scanned across said surface of said substrate at a speed of at least about 30 image lines/second.