Fluorescence detection type electrophoresis apparatus

• US 5,062,942 A
• Filed: 04/10/1990
• Issued: 11/05/1991
• Est. Priority Date: 04/12/1989
• Status: Expired due to Term

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Sumitani, Tomoaki, Ito, Yoshitoshi, Kambara, Hideki, Nishikawa, Tetsuo, Nagai, Keiichi
• Primary Examiner(s): Tung, T.
• Assistant Examiner(s): RYSER, DAVID
• Application Number:
  US07/506,986
• Time in Patent Office: 574 Days
• Field of Search: 204/180.1, 204/182.8, 204/299 R, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 356/344
• US Class Current: 204/612
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6469 Cavity, e.g. ellipsoid
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

In a fluorescence detection type electrophoresis apparatus, comprising an electrophoresis separation gel plate (2, 102, 205), an excitation laser beam source (3, 201, 201) for emitting fluorescent light, and a fluorescent light detector (9, 109, 212) for detecting the emitted fluorescent light, a fluorescent light images are divided into a plurality of virtual images by an image divider (5, 7₂, 104, 105, 209) and at the same time, the lights corresponding to the individual divided images are wavelength-selected by bandpass filters (8, 8₃, 107, 108, 120, 227-230), thereby providing highly accurate, sensitive separation and detection of DNA fragments and the like and determination of base sequence of DNA and the like.

Claims

1. A fluorescence detection type electrophoresis apparatus, comprising:

(i) a migration lane on which sample fragments labeled with fluorophore migrate electrophoretically;

(ii) means for irradiating locally a predetermined portion to be irradiated with an excitation light for exciting said fluorophore, said predetermined portion being on said migration lane;

(iii) optical means for focusing a fluorescent light image which is emitted from said fluorophore at said predetermined irradiated portion, on a plurality of focusing positions separated from each other, said optical means comprising;

a) a focusing lens disposed on a predetermined distance from said predetermined irradiated portion; and

b) an image dividing means disposed on an optical path from said predetermined irradiated portion to said focusing lens and makes said fluorescent light image visible as a plurality of virtual images positionally different from each other when said light image is seen from the focusing lens side;

iv) a plurality of wavelength selection means disposed on the respective optical paths from said predetermined irradiated portion to said plurality of focusing positions, said respective optical paths lying separately from each other at the position of said wavelength selection means, and said plurality of wavelength selection means having transmission wavelength band different from each other; and

v) a light detector for detecting with positional distinction each light amount of a plurality of fluorescent light images each of which had passed through each of said wavelength selection means and focused on each of said focusing positions.

2. A fluorescence detection type electrophoresis apparatus according to claim 1, in which the light detector is a two-dimensional detector.

3. A fluorescence detection type electrophoresis apparatus according to claim 1, which separates and detects DNAs or RNAs.

4. A fluorescent detection type electrophoresis apparatus comprising:

i) a platelike migration lane on which a plurality of sample fragments migrate electrophoretically, said plurality of sample fragments being labeled respectively with a plurality of fluorophores having different maximum emission wavelengths from each other;

ii) means for irradiating locally a predetermined portion to be irradiated with at least one laser beam for exciting said plurality of fluorophores, said predetermined portion being on said migration lane and having a bandlike shape with a longitudinal side perpendicular to the migration direction of said fragments;

iii) optical means for focusing a bandlike fluorescent light image, which is emitted from the fluorophore at said predetermined irradiated portion, on a plurality of focusing positions separated from each other, said optical means comprising;

a) a focusing lens disposed on a predetermined distance from said predetermined irradiated portion; and

b) an image dividing means which is disposed on an optical path from said predetermined irradiated portion to said focusing lens and which makes said fluorescent light image visible as a plurality of virtual images positionally different from each other when said light image is seen from the focusing lens side;

iv) a plurality of band-pass filters disposed on the respective optical paths from said predetermined irradiated portion to said plurality of focusing positions, said respective optical paths lying separately from each other at the positions of said band-pass filters, and each of said plurality of band-pass filters selecting a fluorescent light emitted from one of said fluorophores from the other fluorescent lights emitted from the other fluorophores with respect to the wavelength and transmitting the selected fluorescent light; and

v) a light detector for detecting with positional distinction each light amount of a plurality of band-like fluorescent light images which has passed through each of said band-pass filters and focused on each of said focusing positions, to thereby detect each of said plurality of sample fragments in passing through said predetermined irradiated portion.

5. A fluorescence detection type electrophoresis apparatus according to claim 4, in which said platelike migration lane is formed in a slab gel which is disposed between a pair of electrophoresis separation gel plates, the laser beam irradiation on said predetermined portion is directed from a side end surface side of the gel plates and penetrated through said slab gel of the electrophoresis separation gel plates, which is positioned in parallel with the electrophoresis separation gel plates, and a fluorescent light line image obtained by the laser irradiation is emitted along the path of the laser beam in the slab gel.

6. A fluorescence detection type electrophoresis apparatus according to claim 4, in which the image dividing means comprises a plurality of reflection mirrors arranged in parallel with an axis of a laser beam in the electrophoresis separation gel plates and in which a corresponding number of bandpass filters of different transmission wavelengths is provided on respective paths leading the fluorescent lights reflected by the reflection mirrors to focusing positions on said light detector which is a fluorescent light detector.

7. A fluorescence detection type electrophoresis apparatus according to claim 6, in which the plurality of the reflection mirrors are arranged on circumference of an ellipse having its center lying on said predetermined portion.

8. A fluorescence detection type electrophoresis apparatus according to claim 4, in which the image dividing means comprises at least a plurality if lenses of different optic axes or a lens divided into fragments of different optic axes said image dividing means being provided on a path on which fluorescent lights emitted from said predetermined portion propagate to the focusing position on the light detector.

9. A fluorescence detection type electrophoresis apparatus according to claim 8, in which an additional prism or mirror for dividing the fluorescent light image is provided on a path on which fluorescent lights emitted form said predetermined portion propagate to the focusing position on the light detector.

10. A fluorescent detection type electrophoresis apparatus comprising:

i) a platelike migration lane on which sample fragments labeled with fluorophore migrate electrophoretically;

ii) means for irradiating locally a predetermined portion to be irradiated with a least one laser beam for exciting said fluorophores, said predetermined portion being on said migration lane and having a bandlike shape with a longitudinal side perpendicular to the migration direction of said fragments;

iii) a prism having an incidence plane on which the fluorescent light emitted from said fluorophore at the predetermined irradiated portion is made to be incident and having a plurality of light-outgoing planes;

iv) a focusing lens which is disposed at a position capable of viewing said predetermined irradiated portion though said plurality of light-outgoing planes of said prism and by which said fluorescent lights outgoing from said plurality of light out-going planes focus respectively on focusing positions different from each other to thereby form a plurality of respective bandlike image;

v) a plurality of wavelength selection means disposed on the respective optical paths from said predetermined irradiated portion to said plurality of focusing positions, said respective optical paths lying separately from each other at the position of said wavelength selection means, and said plurality of wavelength selection means having transmission wavelength bands different from each other; and

vi) a light detector for detecting with positional distinction each light amount of a plurality of bandlike fluorescent light images each of which has passed through each of said wavelength selection means and focused on each of said focusing positions.

11. A fluorescent detection type electrophoresis apparatus according to claim 10, wherein said wavelength selection means is disposed on the respective optical paths and disposed between said focusing lens and said light detector.

12. A fluorescent detection type electrophoresis apparatus according to claim 10, wherein said plurality of wavelength selection means is a multiplane filter having a plurality of light-outgoing planes, which is disposed between said focusing lens and said prism.

13. A fluorescence detection type electrophoresis apparatus according to claim 10, in which said platelike migration lane is formed in a slab gel which is disposed between a pair of electrophoresis separation gel plates, the laser beam irradiation on said predetermined portion is directed from a side end surface side of the gel plates and penetrated through said slab gel of the electrophoresis separation gel plates, which is positioned in parallel with the electrophoresis separation gel plates, and a fluorescent light line image obtained by the laser irradiation is emitted along the path of the laser beam in the slab gel.

14. A fluorescence detection type electrophoresis apparatus according to claim 10, in which the plurality of wavelength selection means is bandpass filters of individually different transmission wavelengths, said bandpass filters being provided on respectively corresponding paths of the divided image lights propagating out of the plurality of the light outgoing planes of the prism.

15. A fluorescence detection type electrophoresis apparatus according to claim 10, in which the fluorescence detection type electrophoresis apparatus is a multicolor fluorescence detection type electrophoresis apparatus, and the separation and detection sample for migrating in the electrophoresis separation plate is a multicolor-labeled sample.

16. A fluorescence detection type electrophoresis apparatus according to claim 10, in which at least one edge of the prism is in a plane including fluorescent light line images obtained as a result of laser beam irradiation to said predetermined portion and a center of the focusing position of the light detector, and is in parallel with the fluorescent light line images.

17. A fluorescence detection type electrophoresis apparatus according to claim 10, in which the prism is symmetric up and down with respect to a plane including fluorescent light line images obtained as a result of laser beam irradiation to said predetermined portion and a center of the focusing position of the light detector, and a vertex of the prism at the center is in parallel with the fluorescent light line images.

18. A fluorescent detection type electrophoresis apparatus comprising:

i) a platelike migration lane on which a plurality of sample fragments migrate electrophoretically, said plurality of sample fragments being labeled respectively with a plurality of fluorophores having different maximum emission wavelengths from each other;

ii) means irradiating locally a predetermined portion to be irradiated with at least one laser beam for exciting said plurality of fluorophores, said predetermined portion being on said migration lane and having a bandlike shape with a longitudinal side perpendicular to the migration direction of said fragments;

iii) a cylindrical convex lens converging a fluorescent light emitted from said fluorophore at said predetermined irradiated portion, in a width direction of said predetermined irradiated portion of bandlike shape;

iv) a prism having an incidence plane on which the fluorescent light transmitted through said cylindrical convex lens is made to be incident and having a plurality of light-outgoing planes;

v) a cylindrical concave lens diverging a fluorescent light outgoing from said plurality of light-outgoing plane of said prism, in a width direction of said predetermined irradiated portion of bandlike shape;

vi) a focusing lens which is disposed at a position capable of viewing said predetermined irradiated portion of bandlike shape through said cylindrical concave lens, said prism and said cylindrical convex lens and which focuses said fluorescent lights outgoing from said plurality of light-outgoing planes respectively on focusing positions different from each other to thereby form respectively a plurality of bandlike images;

vii) a plurality of band-pass filters disposed on the respective optical paths from said predetermined irradiated portion to a plurality of said focusing portions, said respective optical paths lying separately from each other at the positions of said band-pass filters, and each of said plurality of band-pass filters, and each of said plurality of band-pass filters selecting a fluorescent light emitted from one of said fluorophores from the other fluorescent lights emitted from the other fluorophores with respect to the wavelength and transmitting the selected fluorescent light; and

viii) a light detector for detecting with positional distinction each light amount of a plurality of bandlike fluorescent light images which has passed through each of said band-pass filters and focused on each of said focusing positions, to thereby detect each of said plurality of sample fragments in passing through said predetermined irradiated portion.

19. A fluorescence detection type electrophoresis apparatus according to claim 18, in which said platelike migration lane is formed in a slab gel which is disposed between a pair of electrophoresis separation gel plates, the laser beam irradiation on said predetermined portion is directed from a side end surface side of the gel plates and penetrated through said slab gel of the electrophoresis separation gel plates, which is positioned in parallel with the electrophoresis separation gel plates, and a fluorescent light line image obtained by the laser irradiation is emitted along the path of the laser beam in the slab gel.

20. A fluorescence detection type electrophoresis apparatus according to claim 18, in which said plurality of bandpass filters is a divided multiplane filter, by which the fluorescent light image is divided.

21. A fluorescence detection type electrophoresis apparatus according to claim 20, in which the divided multiplane filter is provided between the cylindrical convex lens and said focusing lens.

22. A fluorescence detection type electrophoresis apparatus according to claim 21, in which planes of the divided multiplane filter have areas sized so that amounts of the fluorescent lights passing them can be substantially equal.

23. A fluorescence detection type electrophoresis apparatus according to claim 18, in which the cylindrical convex lens comprises a plurality of divided cylindrical convex lenses, by which the fluorescent light image can be divided.

24. A fluorescence detection type electrophoresis apparatus according to claim 18, in which a slit is provided in parallel with fluorescent light line images between an emitting point of the fluorescent light line images and a cylindrical convex lens to cut out upper and lower parts of the fluorescent lights emitted from the emitting point higher or lower than specific angles.

25. A fluorescence detection type electrophoresis apparatus according to claim 24, in which a width of the slit is made narrower than an interval of virtual object positions.