Arrangement for confocal fluorescence microscopy

• US 5,751,417 A
• Filed: 03/20/1996
• Issued: 05/12/1998
• Est. Priority Date: 03/20/1995
• Status: Expired due to Term

• Current Assignee: TILL Photonics GmbH (Thermo Fisher Scientific Incorporated)
• Original Assignee: Rainer Uhl
• Inventors: Uhl, Rainer
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US08/619,922
• Time in Patent Office: 783 Days
• Field of Search: 356/317, 356/318, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 356/318
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/0205 Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
• CPC Class Codes: G01J 3/0208 using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
• CPC Class Codes: G01J 3/021 using plane or convex mirrors, parallel phase plates, or particular reflectors
• CPC Class Codes: G01J 3/0229 using masks, aperture plates, spatial light modulators or spatial filters, e.g. reflective filters
• CPC Class Codes: G01J 3/14 using refracting elements, e.g. prisms G01J3/18, G01J3/26 take precedence prisms per se G02B5/04
• CPC Class Codes: G02B 21/0064 multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 27/147 using averaging effects by spatially variable reflectivity on a microscopic level, e.g. polka dots, chequered or discontinuous patterns, or rapidly moving surfaces G02B27/1086 takes precedence

Abstract

An arrangement for confocal or quasifocal fluorescence microscopy is suggested in which, in an image beam path, there are arranged in succession, an objective arrangement for acquiring an image of a specimen to be examined, at least one scanner mirror located downstream of the objective arrangement, a tube lens, a confocal first strip diaphragm, a first spectrometer arrangement, a wavelength selection diaphragm for selection of the emission wavelength, a second spectrometer arrangement which is analogous to the first spectrometer arrangement, and a detector for acquiring the brightness distribution. In which, by means of the wavelength selection diaphragm, coupling of excitation light takes place by the reflecting back of an excitation beam path to the wavelength selection diaphragm from a source of monochromatic excitation light by a strip diaphragm which corresponds to the confocal strip diaphragm and a third spectrometer arrangement which is analogous to the first spectrometer arrangement. The image beam path and excitation beam path are constituted and matched to one another such that the light emitted from the specimen reaches the detector by way of the wavelength selection diaphragm. Excitation light from the wavelength selection diaphragm, however, is prevented from hitting the detector, but traverses the confocal strip diaphragm.

Claims

1. An arrangement for confocal and quasifocal fluorescence microscopy in which the following are arranged in succession in an image beam path:

(a) an objective arrangement for acquiring an image of a specimen to be examined;

(b) at least one scanner mirror located below said objective arrangement;

(c) a tube lens for producing an image of said specimen in an image plane;

(d) a first confocal diaphragm located in said image plane of said tube lens;

(e) a first spectrometer arrangement with

(i) a first lens for imaging an opening of said diaphragm at infinity,

(ii) a dispersion prism for spectral decomposition of light emerging from said first lens, and

(iii) a second lens for producing an image in an exit slit plane of said first spectrometer arrangement which represents an intermediate image plane;

(f) a wavelength selection diaphragm located in said exit slit plane with a host of parallel slits with a slit width or spacing which corresponds to a desired bandwidth and which can be shifted perpendicularly to a longitudinal direction of the slits for purposes of selection of an emission wavelength;

(g) a second spectrometer arrangement which is analogous to said first spectrometer arrangement and which forms a subtractive dispersion means to spectrally re-combine the light of said wavelength selection diaphragm which has been spectrally fanned out and to image it in a second image plane; and

(h) a detector for acquiring a brightness distribution in said second image plane;

wherein said wavelength selection diaphragm constitutes a means for coupling of excitation light by an excitation beam path travelling back along an arrangement comprised of said first spectrometer arrangement, said first confocal diaphragm, said tube lens, said scanner mirror and said objective arrangement;

wherein the excitation beam path leads from a source of monochromatic excitation light by way of a second confocal diaphragm, which corresponds to said first confocal diaphragm, and a third spectrometer arrangement, analogous to said first spectrometer arrangement, to said wavelength selection diaphragm;

wherein the image beam path and the excitation beam path are constituted and matched to one another in a manner causing the light emitted from said specimen to reach said detector by way of said wavelength selection diaphragm, and conversely, preventing excitation light from said wavelength selection diaphragm from hitting said detector and causing the excitation light to traverse said first confocal diaphragm and reach said specimen.

2. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 1, wherein said first confocal diaphragm is a confocal strip diaphragm having a plurality of slits in a plane.

3. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein said arrangement further comprises a monochromator with an adjustable wavelength as a source for the monochromatic excitation light.

4. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 3, wherein said monochromator has a white light source and means for selection of spectral components of the light emitted by said white light source for use as said excitation light.

5. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 4, wherein an arc lamp which produces an arc is provided as said white light source and a lens array for using an area of maximum brightness of the arc is assigned to said monochromator.

6. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein a digital mirror device is used as said wavelength selection diaphragm.

7. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein said arrangement further comprises a descanner mirror which is moved analogously to said scanner mirror and which images light strips emitted from the confocal strip diaphragm in time and space coordination with the excitation of said specimen on said detector;

and wherein said detector is an area sensor.

8. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein said detector is designed as a multi-line sensor and an arrangement for descanning of the acquired image information electronically is provided.

9. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 1, wherein said confocal diaphragms are confocal point diaphragms and wherein there are additional scanner and descanner arrangements for scanning and descanning in a direction which runs orthogonally to a scanning direction of said scanner mirror.

10. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 9, wherein said additional scanner arrangement has a piezocrystal for displacement of said objective arrangement.

11. An arrangement for confocal or quasifocal fluorescence microscopy as described in claim 1, wherein the second confocal diaphragm in the excitation beam path is a point diaphragm which can move parallel to the longitudinal direction of the slits of said wavelength selection diaphragm.

12. An arrangement for confocal or quasifocal fluorescence microscopy as described in claim 11, wherein an arrangement which evaluates only the image information which corresponds to the point grid of said point diaphragm can be assigned to said detector.

13. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein said arrangement further comprises an additional source for white observation light to alternatively illuminate said specimen in a transmitted light process.

14. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 2, wherein reflecting cross-pieces of said wavelength selection diaphragm are sloped relative to the plane of the slits of the confocal strip diaphragm.

15. An arrangement for confocal and quasifocal fluorescence microscopy in which the following are arranged in succession in an image beam path:

(a) an objective arrangement for acquiring an image of a specimen to be examined;

(b) at least one scanner mirror located below said objective arrangement;

(c) a dispersion prism for spectral decomposition of the light emerging from said objective arrangement;

(d) a tube lens for producing an image in an intermediate image plane of a first spectrometer arrangement which comprises said objective arrangement, said dispersion prism and said tube lens;

(e) a wavelength selection diaphragm located in said intermediate image plane with a host of parallel slits with a slit or slit spacing which corresponds to the desired bandwidth and which can be shifted perpendicularly to the longitudinal direction of the slits for purposes of selection of the emission wavelength;

(f) a second spectrometer arrangement which is analogous to said first spectrometer arrangement and which forms a subtractive dispersion means to spectrally re-combine the light of said wavelength selection diaphragm which has been spectrally fanned out and to image it in a second image plane; and

(g) a detector for acquiring the brightness distribution in said second image plane;

wherein said wavelength selection diaphragm forms a means for coupling of excitation light by an excitation beam path travelling back along an arrangement which is comprised of said first spectrometer arrangement and said scanner mirror;

the excitation beam path leading from a source for monochromatic excitation light by way of a confocal diaphragm and a third spectrometer arrangement to said wavelength selection diaphragm;

wherein the image beam path and excitation beam path are constituted and matched to one another in a manner causing the light emitted from said specimen to reach said detector by way of said wavelength selection diaphragm and conversely for preventing excitation light from said wavelength selection diaphragm from hitting said detector, and causing the excitation light to traverse said confocal diaphragm and reach said specimen.

16. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 15, wherein said confocal diaphragm is a confocal strip diaphragm.

17. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 16, wherein said arrangement further comprises a monochromator with adjustable wavelength as the source for monochromatic excitation light.

18. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 17, wherein said monochromator has a white light source and means for selection of the spectral components of the light emitted by said white light source which are used as excitation light.

19. An arrangement for confocal and quasifocal fluorescence microscopy as described in claim 15, wherein the confocal diaphragm in the excitation beam path is a point diaphragm which is movable parallel to the longitudinal direction of the slits of said wavelength selection diaphragm.

20. A confocal fluorescence microscope with a specimen to be examined, a monochromatic excitation light source, a first and a second strip diaphragm, a first prism spectrometer, a second prism spectrometer which is analogous to said first prism spectrometer, a third prism spectrometer and a detector for the fluorescent light emitted from said specimen;

the excitation light being incident on a scanner mirror and from there on said specimen through an objective arrangement with focus on said specimen and the fluorescent light of said specimen in a reverse direction reaching said detector through said objective arrangement by means of said scanner mirror, in which said first and said third prism spectrometers are attached in an excitation light path;

said third prism spectrometer following said second strip diaphragm with a host of parallel slits; and

said first prism spectrometer being followed by said first strip diaphragm which is located confocally to said objective arrangement and which is identical to said second strip diaphragm;

said third prism spectrometer forming a means for fanning out the excitation light emerging from said second strip diaphragm and focusing it in an intermediate image plane, from where the fanned-out excitation light is incident in said first prism spectrometer, is concentrated by the latter, and is focussed on said first strip diaphragm;

said first prism spectrometer forming a means for fanning out the fluorescent light emerging from said first strip diaphragm in said detector direction and focusing it in said intermediate image plane from where the fanned-out fluorescent light is incident in said second prism spectrometer and said third prism spectrometer and is concentrated by said second prism spectrometer and is focussed on said detector;

wherein a selection diaphragm which separates the fluorescent light from the excitation light is provided in said intermediate image plane and is formed as a strip diaphragm with a host of parallel slits, with reflecting cross-pieces in between said parallel slits, and which are aligned parallel to said slits of said first and said second strip diaphragm and which are dimensionally configured and positionally arranged with respect to them, such that either said cross-pieces reflect the excitation light to said specimen and the fluorescent light is incident in a straight line through said slits of said selection diaphragm on said detector, or conversely the excitation light is incident in a straight line through said slits of said selection diaphragm on said specimen and said cross-pieces reflect the fluorescent light towards said detector; and

wherein said selection diaphragm is movable for purposes of adjustment to a desired fluorescent wavelength perpendicularly to the longitudinal direction of said slits.

21. A confocal fluorescence microscope as described in claim 20, wherein said microscope further comprises a monochromator with an adjustable wavelength as the source for monochromatic excitation light.

22. A confocal fluorescence microscope as described in claim 21, wherein said monochromator has a white light source and means for selection of the spectral components of the light emitted by said white light source which are used as excitation light.