Apparatus and method for confocal observation of a sample
A device for confocal observation of a sample comprises at least one disk-like body, wherein the at least one disk-like body comprises on a first side a plurality of micro-optical elements and on a second side a correspondingly arranged pinhole pattern formed by a plurality of pinholes, wherein the plurality of micro-optical elements arranged on the first side comprise a convex, dichroically coated front surface, wherein for excitation of the sample the disk is illuminated on its side facing the tube lens with collimated light which is reflected back to the tube lens by the plurality of curved, dichroically coated surfaces on the disk in such a way that a corresponding excitation spot pattern is created therefrom in the object plane of the microscope, which excitation spot pattern, after having passed the tube lens and the plurality of curved, dichroically coated surfaces, can be spatially filtered and detected.
1 . A structured disk for confocal observation of a sample with an imaging optical system, comprising:
a single disk-like body having a plurality of micro-optical elements on a first side and a correspondingly arranged pinhole pattern on a second side, the correspondingly arranged pinhole pattern formed by a plurality of pinholes, and each of the plurality of micro-optical elements arranged on the first side having a convex, dichroically coated front surface, wherein
a distance between the first side and the second side of the single disk-like body is selected such that light which is inverse to divergent light resulting from reflection of collimated light at the plurality of micro-optical elements on the first side is focused into respective pinholes after transmission through the dichroic coating.
2 . The structured disk according to claim 1 , wherein the convex, dichroically coated front surfaces are molded in a medium having a same refractive index as the plurality of micro-optical elements and thus having an uncurved surface arranged parallel to the single disk-like body, wherein a plane of virtual illumination spots coincides with a plane of a pinhole mask.
3 . The structured disk according to claim 1 , wherein the convex, dichroically coated front surfaces on the first side are opposed by a second arrangement of convexly curved surfaces on an opposite side.
4 . The structured disk according to claim 1 , wherein the single disk-like body is formed of glass.
5 . A structured disk for confocal observation of a sample with an imaging optical system, comprising:
at least one disk-like body having a plurality of micro-optical elements on a first side and a correspondingly arranged pinhole pattern on a second side, the correspondingly arranged pinhole pattern formed by a plurality of pinholes, and each of the plurality of micro-optical elements arranged on the first side having a convex, dichroically coated front surface, wherein
the convex, dichroically coated front surfaces are molded in a medium having a same refractive index as the plurality of micro-optical elements and thus having an uncurved surface arranged parallel to the at least one disk-like body,
a plane of virtual illumination spots coincides with a plane of a pinhole mask,
the at least one disk-like body includes a first disk-shaped body and a second disk-shaped body, and
the plurality of micro-optical elements on the first disk-shaped body and the plurality of pinholes on the second disk-shaped body are arranged in correlation with each other.
6 . The structured disk according to claim 5 , wherein the second disk-shaped body has a plurality of conjugate diverging lenses arranged thereon, each conjugate diverging lens being arranged correspondingly with a micro-optical element and a pinhole about respective central axes.
7 . The structured disk according to claim 5 , wherein the at least one disk-like body is formed of glass.
8 . A device for confocal observation of a fluorescent sample with a microscope, the device comprising:
a disk for confocal observation of a sample with an imaging optical system, the disk including at least one disk-like body, the at least one disk-like body having a plurality of micro-optical elements on a first side and a correspondingly arranged pinhole pattern on a second side, the correspondingly arranged pinhole pattern formed by a plurality of pinholes, and each of the plurality of micro-optical elements arranged on the first side having a convex, dichroically coated front surface; wherein
the disk is structured about a central axis, arranged between a tube lens and a detector of the microscope, illuminated with collimated light for excitation on a side facing the tube lens,
the collimated light, due to the convex, dichroically coated front surfaces on the disk, produces a virtual spot pattern behind the convex, dichroically coated front surfaces and reflects rays back to the tube lens such that a corresponding excitation spot pattern is created therefrom in an object plane of the microscope, the corresponding excitation spot pattern generating a fluorescent spot pattern, which, after passing backwards through the tube lens, hits again the convex, dichroically coated front surfaces where excitation light was reflected, but is now transmitted due to a dichroic property of the convex, dichroically coated front surfaces, and
a distance between the first side of the disk and the second side of the disk is selected such that ray bundles of the transmitted fluorescent spot pattern combine to form focal points in a plane of the plurality of pinholes and can be spatially (confocally) filtered by the correspondingly arranged pinhole pattern.
9 . The device according to claim 8 , further comprising:
a coupling mirror arranged centrally in a focal plane of the tube lens, wherein
the coupling mirror is aligned in an image beam path such that light focused onto the coupling mirror by a focusing element hits the plurality of micro-optical elements as collimated excitation light after passing the tube lens and is reflected back to the tube lens.
10 . The device according to claim 9 , wherein the focal plane of the tube lens coincides with a rear focal plane of an objective.
11 . The device according to claim 9 , wherein
the disk, which is rotatable about the central axis, is replaced by a planar dichroic mirror, the planar dichroic mirror being tilted by an angle, and
the reflected excitation light in the focal plane of the tube lens, which coincides with a focal plane of an objective, is deflected past the coupling mirror and formed by the objective into an oblique wide-field illumination of the sample.
12 . The device according to claim 8 , wherein the at least one disk-like body is formed of glass.
13 . A method for confocal observation of a fluorescent sample with a microscope, the method comprising:
illuminating with collimated light a plurality of curved, dichroically mirrored surfaces on a structured disk arranged between a first tube lens and a detector of a microscope according to claim 1 , wherein a virtual spot pattern is produced behind the plurality of curved, dichroically mirrored surfaces and rays are reflected back to the first tube lens such that a corresponding excitation spot pattern is created therefrom in an object plane of the microscope, wherein
the corresponding excitation spot pattern generates a fluorescent spot pattern, which, after passing backwards through the first tube lens, hits again the plurality of curved, dichroically mirrored surfaces where excitation light was reflected, but is now transmitted due to a dichroic property of the plurality of curved, dichroically mirrored surfaces, and
a distance between the first side of the structured disk and the second side of the structured disk is selected such that ray bundles of the transmitted fluorescent spot pattern combine to form focal points in a plane of the plurality of pinholes and can be spatially (confocally) filtered by the correspondingly arranged pinhole pattern; and
detecting a confocally filtered fluorescent spot pattern with the detector.
14 . The method according to claim 13 , further comprising:
coupling collimated excitation light via a coupling mirror arranged centrally in a focal plane of the first tube lens, wherein
the coupling mirror is aligned in an image beam path such that light focused onto the coupling mirror by a second tube lens, after passing the first tube lens, hits the plurality of curved, dichroically mirrored surfaces as collimated excitation light and is reflected back to the first tube lens.