Method and device for determining the location and number of fluorescent molecules

• US 5,528,046 A
• Filed: 11/10/1993
• Issued: 06/18/1996
• Est. Priority Date: 11/10/1992
• Status: Expired due to Fees

• Current Assignee: Hamamatsu Photonics KK
• Original Assignee: Hamamatsu Photonics KK
• Inventors: Ishikawa, Mitsuru
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/149,840
• Time in Patent Office: 951 Days
• Field of Search: 250/461.2
• US Class Current: 250/461.2
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6402 Atomic fluorescence; Laser induced 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

The present invention is directed to a device and method for determining the presence of fluorescent molecules, locations thereof, and number of fluorescent molecules in a local area irradiated with excitation light based on the number of fluorescence photons generated per unit period of time by the irradiation of the excitation light counted as a quantized fluorescence intensity of the fluorescence molecules and a distribution of appearance frequencies of the fluorescence photons formed on a two-dimensional image. In accordance with the present invention, a device is provided which comprises a light source for irradiating excitation light to a local area on a flat substrate with the adsorbed fluorescent molecules, and fluorescence detecting means for detecting fluorescence photons from the fluorescent molecules and for forming a distribution of appearance frequencies of the fluorescence photons per unit period of time on a two-dimensional image corresponding to the local area.

Claims

1. A device for determining a location of a molecule-group and the number of fluorescent molecules in said molecule-group comprising:

a light source for irradiating a local area on a flat substrate with excitation light, said local area having a molecule-group comprising an adsorbed fluorescent molecule; and

fluorescence detecting means for detecting fluorescence photons generated by irradiating said local area on said substrate with excitation light, for identifying a fluorescent spot where said molecule-group is present based on a two-dimensional frequency distribution of said fluorescence photons, and for determining said number of said fluorescence molecules in said molecule-group by measuring the number of said fluorescence photons per unit period of time as a quantized fluorescence intensity of said molecule-group, measured in said fluorescent spot.

2. A device according to claim 1, wherein

said fluorescence detecting means is disposed at a position which is out of an optical path of said excitation light irradiated from said light source and reflected light from a surface of said substrate.

3. A device according to claim 2, wherein

said fluorescence detecting means includes an optical microscope for condensing said fluorescence in said local area on said surface of said substrate;

and

a photon counting system for counting said number of said fluorescence photons condensed by said optical microscope, and displaying an appearance frequency distribution of said fluorescence photons on a two-dimensional image corresponding to said local area.

4. A device according to claim 3, wherein:

said photon counting system forms an appearance frequency distribution of said fluorescence photons for said unit period of time by plotting a total number of fluorescence photons corresponding to a measuring position of said fluorescent photons on a two-dimensional image corresponding to said local area on said substrate, every time said fluorescence photons are detected by irradiating said excitation light; and

said photon counting system identifies a fluorescent spot where said molecule-group is present, based on said appearance frequency distribution and determines said number of said fluorescent molecules in said molecule-group based on said number of said fluorescent photons measured in said fluorescent spot.

5. A device according to claim 3, wherein

said optical microscope can change its magnification.

6. A device according to claim 2, wherein

said excitation light includes a laser beam.

7. A device according to claim 1,

further comprising moving means for relatively displacing said local area on said substrate.

8. A device according to claim 7, wherein

said moving means includes an X-Y stage being movable horizontally to said fluorescence detecting means, and a rotary stage supported by said X-Y stage for fixing said substrate.

9. A device for determining a sequence of bases in nucleic acid comprising:

a light source for irradiating a a local area having a base therein with excitation light, said local area being on a flat substrate holding a sequence of said bases of nucleic acid;

fluorescence detecting means for detecting fluorescence generated from said base present in said local area on said flat substrate and for identifying a kind of said base based on a wavelength distribution of said fluorescence and a lifetime of said fluorescence;

moving means for moving said substrate; and

means for controlling said moving means so as to relatively move said local area on said flat substrate along said sequence of bases.

10. A device according to claim 9, wherein

said fluorescence detecting means includes an optical microscope for condensing said fluorescence in said local area on said surface of said substrate;

and

a photon counting system for counting the number of fluorescence photons condensed by said optical microscope and displaying an appearance frequency distribution of said fluorescence photons on a two-dimensional image corresponding to said local area.

11. A device according to claim 9, wherein

said fluorescence detecting means is disposed at a position which is out of an optical path of said excitation light irradiated by said light source and of a reflected light from said surface of said substrate.

12. A device according to claim 11, wherein

said light source includes a laser device.

13. A device according to claim 9, wherein

said substrate includes a silicon wafer.

14. A device according to claim 13, wherein

said substrate is disk-shaped.

15. A device according to claim 9, wherein

said moving means includes an X-Y stage being movable horizontally to said fluorescence detecting means, and a rotary stage supported by said X-Y stage for fixing said substrate.

16. A device according to claim 9, comprises cooling means for cooling said local area on said surface of the substrate.

17. A device for determining a sequence of bases in nucleic acid comprising:

a light source for irradiating a local area having a base therein with excitation light, said local area being on a flat substrate holding a sequence of said bases of nucleic acid;

fluorescence detecting means for detecting fluorescence generated from said base present in said local area on said flat substrate and for identifying a kind of said base based on a wavelength distribution of said fluorescence and a lifetime of said fluorescence;

moving means for respectively moving said local area on said flat substrate along said sequence of said bases; and

cooling means for cooling said fluorescent molecule in said local area of said substrate, wherein said substrate is positioned between said fluorescence detecting means and said cooling means.