Apparatus and method for the rapid spectral resolution of confocal images

• US 6,134,002 A
• Filed: 01/14/1999
• Issued: 10/17/2000
• Est. Priority Date: 01/14/1999
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Simon, John D., Stimson, Michael J.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): LAUCHMAN, LAYLA G
• Application Number:
  US09/229,874
• Time in Patent Office: 642 Days
• Field of Search: 356/326, 356/346, 356/318, 356/319, 356/72, 359/368, 250/458.1, 250/459.1, 250/461.1, 250/235
• US Class Current: 356/326
• CPC Class Codes: G01J 3/2803 using photoelectric array detector
• CPC Class Codes: G01J 3/2823 Imaging spectrometer
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0056 based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

A confocal scanning microscope apparatus and method is used to rapidly acquire spectrally resolved images. The confocal scanning microscope apparatus includes optics used to simultaneously acquire at least two points along a scan pattern on a sample plane of a sample, wherein the points include regions of the sample represented by at least two pixels. A detection arm is placed in the path of the light reflected, scattered, or emitted from the sample plane comprising a spectrometer having a slit for receiving such light and a detector array placed behind the spectrometer. The image corresponding to the at least two points is recorded on the first axis of said detector and the spectral resolution thereof is simultaneously recorded on the second axis of said detector. The confocal scanning microscope apparatus and method offers significant improvements to the current techniques for genetic sequencing, as well as significant improvements to traditional confocal scanning microscope applications.

Claims

1. A confocal scanning beam microscope, comprising:

a) means for supporting a sample to be observed and measured;

b) an illumination source producing a light beam along a path toward said sample;

c) means for focusing the light beam on a prescribed sample plane of a sample;

d) means for scanning the light beam in a predetermined scan pattern on said sample plane;

e) means for simultaneously acquiring at least two points of said predetermined scan pattern on the sample plane, wherein said points include a region of the sample represented by at least two image pixels;

f) a detection arm placed in the path of light reflected, scattered, or emitted from said region on said sample plane of said sample, comprising means for resolving the spectra of said light from each said region including a light receiving opening, and a detector array placed behind said means for spectrally resolving said region;

g) means for focusing the light from said region reflected, scattered, or emitted from said sample plane of said sample to said light receiving opening of said means for resolving the spectra of the light from said region; and

h) wherein said detector array comprises means for detecting the image of said region and means for simultaneously detecting the spectra of said light of said region.

2. A confocal scanning beam microscope as recited in claim 1, further comprising means for descanning light reflected, scattered, or emitted from at least one region of said sample plane.

3. A confocal scanning beam microscope as recited in claim 2, wherein said detection arm further comprising means for resolving the spectra of said descanned light from each said region.

4. A confocal scanning beam microscope as recited in claim 3, further comprising means for focusing said region to said light receiving opening of means for resolving the spectra of said descanned light from each said region.

5. A confocal scanning beam microscope as recited in claim 4, wherein said detector array comprises means for detecting the image of said region and means for simultaneously detecting the spectra of said descanned light of said region.

6. A confocal scanning beam microscope as recited in claim 1, wherein said confocal microscope is of the point-scanning type.

7. A confocal scanning beam microscope as recited in claim 1, wherein said confocal microscope if of the line-scanning type.

8. A confocal scanning beam microscope as recited in claim 1, wherein said means for scanning the light beam in a predetermined scan pattern on said sample plane further comprises a first scan mirror scanning said light beam in a first axis and directing said light beam to a second scan mirror, wherein said second scan mirror scanning said light beam in a second axis to form a raster pattern on said sample plane of the sample and partially descanning the light reflected, scattered, or emitted from at least one region of said sample plane.

9. A confocal scanning beam microscope as recited in claim 8, wherein said detection arm is located for receiving said partially descanned light and focusing it upon said light receiving opening.

10. A confocal scanning beam microscope as recited in claim 9, wherein said first scan mirror scans fast along said first axis and said second scan mirror scans slow along said second axis such that said first axis is projected onto said light receiving opening.

11. A confocal scanning beam microscope as recited in claim 10, said detection arm further comprising a two dimensional detector having first and second axes, wherein said image of said region is recorded on said first axis of said detector and said spectral resolution is simultaneously recorded on said second axis of said detector.

12. A confocal scanning beam microscope as recited in claim 1, wherein a plurality of microcapillaries contain said samples arranged in a coplanar parallel array located within said sample plane.

13. A confocal scanning beam microscope as recited in claim 1, wherein a solgel contains said samples located within said sample plane.

14. A confocal scanning beam microscope as recited in claim 1, wherein an electrophoretic gel contains said samples located within said sample plane.

15. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of fluorescent samples located within said sample plane.

16. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of biomolecules.

17. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of DNA.

18. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of a cellular system.

19. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of isolated cells.

20. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of organelles.

21. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of antibodies.

22. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of encapsulated molecules.

23. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of biochips.

24. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised of polymers.

25. A confocal scanning beam microscope as recited in claim 1, wherein said sample to be observed is comprised ligand crystals.

26. A confocal scanning beam microscope as recited in claim 1, wherein said light receiving opening of said means for spectrally resolving each said region is formed as a slit.

27. A method for rapid acquisition of spectrally resolved confocal images comprising:

a) supporting a sample to be observed and measured;

b) transmitting a light beam along a path toward said sample;

c) focusing the light beam on a prescribed sample plane of a sample;

d) scanning the light beam in a predetermined scan pattern on said sample plane;

e) acquiring simultaneously at least two points of said predetermined predetermined scan pattern on the sample plane, wherein said points include a region of the sample represented by at least two image pixels;

f) focusing said region to said light receiving opening of means for resolving the spectra of the light from each said region; and

g) recording simultaneously the image of said region along one axis of a detector and spectral resolution of the image of said region along a second axis of said detector.

28. A method for rapid acquisition of spectrally resolved confocal images as recited in claim 27 further comprising:

a) descanning light reflected, scattered, or emitted from at least one region of said sample plane;

b) resolving the spectral of said descanned light from each said region;

c) focusing said region to said light receiving opening of means for resolving the spectra of said descanned light from each said region;

d) recording said image of each said region on the first axis of said detector; and

e) recording, simultaneous with recording said image of said region on the first axis of said detector, the spectral resolution of said image of said region on the second axis of said detector.

29. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescence labeled separated samples contained in a plurality of microcapillaries, comprising:

a) means for supporting said samples to be observed and measured;

b) an illumination source producing a light beam along a path toward said sample;

c) means for focusing the light beam on a prescribed sample plane of said samples, wherein said samples are separated in a plurality of microcapillaries arranged in a coplanar parallel array located within said sample plane;

d) means for scanning the light beam in a predetermined scan pattern on said sample plane;

e) means for simultaneously acquiring a line of points of said predetermined scan pattern on the sample plane, wherein said line of points run perpendicular to and across said plurality of microcapillaries;

f) a detection arm placed in the path of light reflected, scattered, or emitted from said line of points on said sample plane of said samples, comprising means for resolving the spectra of said line of points including a light receiving opening, and a detector array placed behind said means for spectrally resolving said line of points;

g) means for focusing the light from said line of points reflected, scattered, or emitted from said sample plane of said samples to said light receiving opening of means for resolving the spectra of the light from each said region; and

h) wherein said detector arm comprises means for detecting the image of said line of points and means for simultaneously detecting the spectra of said light of said line of points.

30. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 29, further comprising means for descanning light reflected, scattered, or emitted from said line of points of said sample plane.

31. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 30, wherein said detection arm further comprising means for resolving the spectra of said descanned light from said line of points of said sample plane.

32. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 31, further comprising means for focusing said region to said light receiving opening of means for resolving the spectra of said descanned light from said line of points of said sample plane.

33. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 32, wherein said detector array comprises means for detecting the image of said line of points and means for simultaneously detecting the spectra of said descanned light of said line of points.

34. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 29, wherein said confocal microscope is of the point-scanning type.

35. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 29, wherein said confocal microscope is of the line-scanning type.

36. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 29, wherein said means for scanning the light beam in a predetermined scan pattern on said sample plane further comprises a first scan mirror scanning said light beam in a first axis and directing said light beam to a second scan mirror, wherein said second scan mirror scanning said light beam in a second axis to form a raster pattern on said sample plane of the sample and partially descanning the light reflected, scattered, or emitted from at least one line of points of said sample plane.

37. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 29, wherein said detection arm is located for receiving said partially descanned light and focusing it upon said light receiving opening.

38. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 36, wherein said first scan mirror scans fast along said first axis and said second scan mirror scans slow along said second axis such that said first axis is projected onto said light receiving opening.

39. A confocal scanning beam microscope for scanning a gel to detect fluorescence from fluorescently labeled separated samples contained in a plurality of microcapillaries as recited in claim 31, said detection arm further comprising a two dimensional detector having first and second axes, wherein said image of said line of points of said sample plane is recorded on said first axis of said detector and said spectral resolution is simultaneously recorded on said second axis of said detector.