In-line complete spectral fluorescent imaging of nucleic acid molecules

• US 20020009744A1
• Filed: 05/18/2001
• Published: 01/24/2002
• Est. Priority Date: 08/18/1998
• Status: Active Grant

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Valery Bogdanov
• Inventors: Bogdanov, Valery
• Granted Patent: US 6,495,363 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2021/6423 Spectral mapping, video display
• 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 a hyperspectral imaging apparatus and methods for employing such an apparatus for multi-dye/base detection of a nucleic acid molecule coupled to a solid surface.

Claims

7. A method for hyperspectral imaging a fluorescently labeled nucleotide analog, wherein said method comprises the steps:

(a) emitting a transmission beam from a light source;

(b) expanding said transmission beam by passing said transmission beam through an expansion lens;

(c) focusing said expanded transmission beam into a focus line by passing said expanded transmission beam through a focusing lens;

(d) contacting said fluorescently labeled nucleotide analog with said focused transmission beam, wherein said contact between said focused transmission line and said fluorescently labeled nucleotide analog excites said fluorescently labeled nucleotide analog to emit a fluorescent emission;

(e) collecting said fluorescent emission with a collection lens;

(f) projecting said collected fluorescent emission into an imaging spectrometer;

(g) detecting said projected fluorescent emission using a detector.

1. A hyperspectral fluorescent imaging apparatus for microarray detection which comprises:

(a) a light source, wherein said light source is capable of emitting a transmission beam;

(b) an expansion lens;

(c) a focusing lens, wherein said focusing lens focuses said transmission beam into a thin focus line;

(d) a collection lens;

(e) an imaging spectrometer; and

(f) a detector.

2. The hyperspectral fluorescent imaging apparatus of claim 1, wherein said apparatus further comprises a translation stage.

3. The hyperspectral fluorescent imaging apparatus of claim 1, wherein said apparatus comprises a first expansion lens and a second expansion lens.

4. The hyperspectral fluorescent imaging apparatus of claim 1, wherein said detector is a charged coupled device.

5. The hyperspectral fluorescent imaging apparatus of claim 1, wherein said light source is a laser.

6. The hyperspectral fluorescent imaging apparatus of claim 5, wherein said laser is a CW Ar laser.

8. The method of claim 7, wherein said fluorescently labeled nucleotide analog is incorporated into a sequencing reagent in a polymerase-meditated, template-dependent reaction.

9. The method of claim 8, wherein said sequencing reagent is immobilized to a solid surface.

10. The method of claim 9, wherein said immobilized sequencing reagent is immobilized as an array.

11. The method of claim 9, wherein said immobilized sequencing reagent comprise a plurality of distinct sequencing reagents.

12. The method of claim 9, wherein said sequencing reagent is selected from the group consisting of a DNA molecule, a RNA molecule, and a PNA molecule.

13. The method of claim 12, wherein said DNA molecule is a cDNA molecule or fragment thereof.

14. The method of claim 13, wherein said cDNA molecule is an EST molecule.

15. The method of claim 8, wherein said polymerase-mediated, template-dependent reaction is Genetic Bit Analysis.

16. The method of claim 9, wherein said solid surface is a glass surface.

17. The method of claim 16, wherein said glass surface is selected from the group consisting of a microscope slide, a coverslip, a glass plate and a bead.

18. The method of claim 7, wherein said light source is a laser.

19. The method of claim 18, wherein said laser is an CW Ar laser.

20. The method of claim 7, wherein said detector is a charged coupled device.

11-1. The method of claim 10, wherein said array is a nested array.