# US 2013/0155218 A1 · High Resolution Microscope and Image Divider Assembly

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- Number: US 2013/0155218 A1
- Document ID: US20130155218A1
- Status: Published application
- Kind: A1
- Application number: 13/518,108
- Filed: 2010-12-14
- Published: 2013-06-20
- Earliest claimed: 2009-12-22
- Assignees: Carl Zeiss Microscopy GmbH
- Inventors: Dr. Thomas Kalkbrenner; Michael Goelles
- Web page: https://patentgrep.com/patent/US20130155218A1

## CPC

- G02B 21/361: {Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 17/023: {for extending or folding an optical path, e.g. delay lines}
- G02B 21/0032: {Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06)}
- G02B 21/0072: {details concerning resolution or correction, including general design of CSOM objectives}
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G02B 21/18: Arrangements with more than one light path, e.g. for comparing two specimens
- G02B 21/367: {providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison}
- G02B 27/106: {for splitting or combining a plurality of identical beams or images, e.g. image replication}
- G02B 27/1066: {for enhancing image performance, like resolution, pixel numbers, dual magnifications or dynamic range, by tiling, slicing or overlapping fields of view}
- G02B 27/141: {using dichroic mirrors}
- G02B 27/144: {using partially transparent surfaces without spectral selectivity (G02B27/147 takes precedence)}
- G02B 27/145: {having sequential partially reflecting surfaces}
- G02B 27/58: Optics for apodization or superresolution; Optical synthetic aperture systems
- G02B 7/38: measured at different points on the optical axis {, e.g. focussing on two or more planes and comparing image data}
- G02B 21/0088: {Inverse microscopes}
- G02B 21/245: {using auxiliary sources, detectors}
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to a microscope having an illumination beam path with wide field illumination of a sample and a first detection beam path having a spatially resolved surface receiver, which is reached by a first part of the detection light coming from the sample via the first detection beam path, or an image divider assembly for a microscope. In order to lengthen the optical path length, at least a second part of the detection light coming from the sample is masked out of the detection beam path and, via deflection means belonging to the detection beam path, is led into a second detection beam path and, preferably via further deflection means, is deflected back in the direction of the detection in such a way that detection light is applied to at least two partial regions beside one another on the surface receiver. At least the second part of the detection light runs in an optical element having an optical density that is increased as compared with the first detection beam path, in order to lengthen the optical path length, and the optical element is designed to be displaceable at an angle, preferably perpendicular, to the optical axis of the first detection beam path in order to adjust the optical path length, and has flat surfaces, at least on the light entry and light exit side thereof; a prism is provided, preferably a glass prism, preferably at least in the second detection beam path after a first beam deflection, for deflection in a direction parallel to the first detection beam path, in order to increase the path length and for reverse deflection.

## Claims

### Claim 1

A microscope having an illumination beam path with wide-field illumination of a sample and a first detection-beam path that includes a spatially resolved surface receiver, that is reached by a first part of the detection light coming from the sample via the first detection-beam path or an image splitter arrangement for a microscope, and at least a second part of the detection light coming from the sample is masked out of the detection-beam path in order to lengthen the optical path length, and, via deflection means associated with the detection-beam path, it is guided in at least a second detection-beam path and, via further deflection means, is deflected back in the direction of the detection in such a way that detection light is caused to be incident on at least two adjoining partial areas on the surface receiver, wherein at least the second part of the detection light runs at least in part to an optical element having a refractive index that is increased in comparison to the first detection-beam path, in order to lengthen the optical path length, and in that the optical element is designed to be movable at an angle, perpendicular to the optical axis of the first detection beam path, in order to adjust the length of the optical path, and in that it includes flat surfaces at least on its light-entry and light-exit sides.

### Claim 2 (depends on 1)

The microscope according to claim 1, wherein a prism, is provided at least in the second detection beam path after a first beam deflection, for deflection in a direction parallel to the first detection-beam path, in order to lengthen the path length and for reverse deflection.

### Claim 3 (depends on 2)

The microscope according to claim 2, wherein the parallel part of the second detection beam path passes through the prism.

### Claim 4 (depends on 2)

The microscope according to claim 2, wherein the prism is designed to be moved perpendicularly to the optical axis for adjusting the optical path length.

### Claim 5 (depends on 1)

The microscope according to claim 1, further comprising a telecentric beam path from the sample to the detector.

### Claim 6 (depends on 1)

The microscope according to claim 1, wherein the diversion of at least the second detection beam path occurs telecentrically in the direction of the detector plane or is located in a telecentric portion of the detection beam path.

### Claim 7

A microscope having an illumination beam path with wide-field illumination of a sample and a first detection beam path that includes a spatially resolved surface receiver, which is reached by a first part of the detection light coming from the sample via the first detection beam path, or an image splitter arrangement for a microscope, wherein at least a second part of the detection light coming from the sample is diverted out of the detection beam path in order to lengthen the optical path length and, via deflection means associated with the detection-beam path, is guided in at least a second detection beam path and, preferably via further deflection means, is deflected back in the direction of the detection in such a way that detection light is caused to be incident on at least two adjoining partial areas on the surface receiver, and wherein at least one part of the deflection means is designed for being movable at an angle, to the optical axis of the first detection beam path for adjustment of the optical path length.

### Claim 8

A microscope having an illumination beam path with wide-field illumination of a sample and a first detection beam path that includes a spatially resolved surface receiver, which is reached by a first part of the detection light coming from the sample via the first detection beam path, or an image splitter arrangement for a microscope, wherein at least a second part of the detection light coming from the sample is diverted out of the detection beam path in order to lengthen the optical path length and, via deflection means associated with the detection-beam path, it is guided in at least a second detection beam path and, preferably via further deflection means, is deflected back in the direction of the detection in such a way that detection light is caused to be incident on at least two adjoining partial areas on the surface receiver, wherein a splitting takes place into four detection beam paths that impinge upon one or more detectors at an offset to one another via partially transparent mirrors and deflection elements.

### Claim 9 (depends on 8)

The microscope according to claim 8, wherein three partially transparent mirrors and three deflection elements are provided for splitting, and the partially transparent mirrors preferably exhibit splitting ratios of 25/75, 66/33, and 50/50 between transmitted and reflected radiation.

### Claim 10 (depends on 1, 7, 8)

A method for using a high resolution microscope embodying the arrangements according to claim 1, 7 or 8 in a high-resolution microscope, in particular for two- or three-dimensional determination of the position of objects, particularly individual fluorophores, for fluorescent microscopy in high spatial resolution of a sample marked with marking molecules, which can be activated or switched by a signal such that only in the activated or switched state can they be excited to emit fluorescent radiation, wherein the method comprises the following steps:
a) introducing the signal to the sample in such a way that only a subset of the marker molecules present in the sample are activated, with sub-regions existing in the sample in which activated marker molecules have a minimum distance from their nearest neighboring activated marker molecules greater than or equal to a distance that represents a predetermined optical resolution;
b) excitation of the activated molecules to emit fluorescent radiation;
c) detection of fluorescent radiation with a predetermined optical resolution; and
d) generation of an individual image from the luminescent radiation recorded in step 3) wherein the geometric locations of the marker molecules emitting the fluorescent radiation are determined with a spatial resolution enhanced beyond the predetermined optical resolution;
wherein the steps are repeated several times, and the multitude of individual images obtained in this manner are combined to an overall image.

### Claim 11 (depends on 2)

The microscope according to claim 2, wherein said prism is a glass prism.

### Claim 12 (depends on 7)

The microscope according to claim 7, wherein said angle is 90°.