Arrangement for confocal fluorescence microscopy
First Claim
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.
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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.
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Citations
22 Claims
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1. An arrangement for confocal and quasifocal fluorescence microscopy in which the following are arranged in succession in an image beam path:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. An arrangement for confocal and quasifocal fluorescence microscopy in which the following are arranged in succession in an image beam path:
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(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. - View Dependent Claims (16, 17, 18, 19)
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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. - View Dependent Claims (21, 22)
- 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;
Specification