US-12619061-B2 - Microscope and method for autofocusing

US12619061B2US 12619061 B2US12619061 B2US 12619061B2US-12619061-B2

Abstract

According to a method for autofocusing on a microscopic sample, measurement light having a local structure is generated. The measurement light is coupled into the microscope beam path, whereby the measurement light is incident on, and reflected by, the sample. The measurement light reflected by the sample is output from the microscope beam path and split among a plurality of component beam paths which pass over optical paths of different lengths to image the reflected measurement light, whereby a plurality of measurement images assigned to different focus positions on the microscope beam path are obtained. At least the measurement image which comes closest to an ideal image of the local structure of the measurement light is selected. Depending on the focus position assigned to the selected measurement image, a focus position to be used on the microscope beam path for the purpose of microscopic imaging of the sample is set.

Inventors

NILS LANGHOLZ
Markus Sticker
Thomas Nobis

Assignees

CARL ZEISS MICROSCOPY GMBH

Dates

Publication Date: 20260505
Application Date: 20240307
Priority Date: 20230321

Claims (17)

1 . A method for autofocusing on a microscopic sample which is imaged along a microscope beam path, the method comprising the following steps: for the autofocusing, generating measurement light which has a local structure, wherein the local structure of the measurement light is formed by a grid; input coupling the measurement light into the microscope beam path, whereby the measurement light is incident on the sample and reflected by the sample; output coupling the measurement light reflected by the sample from the microscope beam path and splitting it among a plurality of component beam paths which pass over optical paths of different lengths to image the reflected measurement light, whereby a plurality of measurement images assigned to different focus positions on the microscope beam path are obtained; selecting at least the measurement image which comes closest to an ideal image of the local structure of the measurement light; and depending on the focus position assigned to the selected measurement image, setting a focus position to be used on the microscope beam path for the purpose of microscopic imaging of the sample.
2 . The method as claimed in claim 1 , wherein the ideal image images the local structure in focus and/or images the local structure in centered fashion and/or images the local structure symmetrically.
3 . The method as claimed in claim 1 , wherein the measurement light is input coupled into the microscope beam path on an input coupling beam path arranged perpendicularly to the microscope beam path.
4 . The method as claimed in claim 3 , wherein the measurement light reflected by the sample is output coupled from the microscope beam path on an output coupling beam path, the input coupling beam path and the output coupling beam path being located together on one axis.
5 . The method as claimed in claim 1 , wherein the optical paths of different lengths for the component beam paths are brought about by virtue of the measurement light reflected by the sample being split into the component beam paths in different directions, with at least one of the component beam paths being deflected on its optical path.
6 . The method as claimed in claim 1 , wherein the measurement light and a light for illuminating the sample for microscopic imaging of the sample differ in terms of their wavelengths, in terms of their polarizations and/or in terms of their temporal occurrence.
7 . The method as claimed in claim 1 , wherein the measurement light is in the near infrared spectral range of the electromagnetic radiation.
8 . A microscope for examining a sample by microscopy, the microscope is configured for carrying out a method according to claim 1 , comprising the following components: an imaging optical unit for imaging the sample along a microscope beam path; a measurement light source for generating measurement light with a local structure; at least one beam-splitting and/or beam-combining optical component for input coupling the measurement light into the microscope beam path, for output coupling the measurement light reflected by the sample from the microscope beam path and for splitting the measurement light reflected by the sample among a plurality of component beam paths; at least one optical detector for detecting the measurement light reflected by the sample and imaged via the plurality of component beam paths, the component beam paths having optical paths of different lengths to the optical detector; a focus drive for setting a focus position on the microscope beam path; and an electronic autofocus controller serving for automatic control of the focus drive and connected to the optical detector.
9 . The method as claimed in claim 1 , wherein the measurement light and a light for illuminating the sample for microscopic imaging of the sample differ in terms of their wavelengths, in terms of their polarizations and/or in terms of their temporal occurrence.
10 . A method for autofocusing on a microscopic sample which is imaged along a microscope beam path, the method comprising the following steps: for the autofocusing, generating measurement light which has a local structure; input coupling the measurement light into the microscope beam path, whereby the measurement light is incident on the sample and reflected by the sample; output coupling the measurement light reflected by the sample from the microscope beam path and splitting it among a plurality of component beam paths which pass over optical paths of different lengths to image the reflected measurement light, whereby a plurality of measurement images assigned to different focus positions on the microscope beam path are obtained, wherein the measurement light input coupled into the microscope beam path and the measurement light reflected by the sample and entering the microscope beam path are guided according to a triangulation; selecting at least the measurement image which comes closest to an ideal image of the local structure of the measurement light; and depending on the focus position assigned to the selected measurement image, setting a focus position to be used on the microscope beam path for the purpose of microscopic imaging of the sample.
11 . The method as claimed in claim 10 , wherein the local structure of the measurement light is formed by at least one point or by a crescent shape.
12 . A microscope for examining a sample by microscopy, the microscope is configured for carrying out a method according to claim 10 , comprising the following components: an imaging optical unit for imaging the sample along a microscope beam path; a measurement light source for generating measurement light with a local structure; at least one beam-splitting and/or beam-combining optical component for input coupling the measurement light into the microscope beam path, for output coupling the measurement light reflected by the sample from the microscope beam path and for splitting the measurement light reflected by the sample among a plurality of component beam paths; at least one optical detector for detecting the measurement light reflected by the sample and imaged via the plurality of component beam paths, the component beam paths having optical paths of different lengths to the optical detector; a focus drive for setting a focus position on the microscope beam path; and an electronic autofocus controller serving for automatic control of the focus drive and connected to the optical detector.
13 . The method as claimed in claim 10 , wherein the ideal image images the local structure in focus and/or images the local structure in centered fashion and/or images the local structure symmetrically.
14 . The method as claimed in claim 10 , wherein the measurement light is input coupled into the microscope beam path on an input coupling beam path arranged perpendicularly to the microscope beam path.
15 . The method as claimed in claim 14 wherein the measurement light reflected by the sample is output coupled from the microscope beam path on an output coupling beam path, the input coupling beam path and the output coupling beam path being located together on one axis.
16 . The method as claimed in claim 10 , wherein the optical paths of different lengths for the component beam paths are brought about by virtue of the measurement light reflected by the sample being split into the component beam paths in different directions, with at least one of the component beam paths being deflected on its optical path.
17 . The method as claimed in claim 10 , wherein the measurement light is in the near infrared spectral range of the electromagnetic radiation.

Description

BACKGROUND OF THE INVENTION The present invention initially relates to a method for autofocusing on a microscopic sample, which is imaged along a microscope beam path. For this purpose, measurement light which has a local structure is generated. This local structure can be formed by a grid, one or two points, or a crescent shape. Furthermore, the invention relates to a microscope for examining a sample by microscopy. An autofocus is used to automatically focus a camera or any other piece of optical equipment such as a microscope on a subject, for example on a microscopic sample. The prior art has disclosed various methods for hardware-based autofocusing or an autofocus hold, on which a plurality of primary requirements are placed from the user's point of view. Initially, it should be possible to determine and approach a focus position as accurately as possible. The approached focus position should then be maintained as accurately as possible. A capture region for the autofocusing should be as large as possible, which in the case of microscopy means that the greatest possible variation of different coverslip thicknesses can be used, or that it is possible to focus at different sample depths while the focus is maintained. These primary requirements inevitably lead to the conflicting aims of demanding both great accuracy and a large capture region. Allowing for reasonable outlay, it is not possible to simultaneously obtain great accuracy and a large capture region on account of the physical and technical boundary conditions. In addition to the aforementioned primary requirements, secondary requirements are also placed on an autofocusing method. In principle, autofocusing should be implemented quickly. Especially in microscopy, a sample should be exposed to only a small amount of light exposure caused by hardware-based autofocusing. High reproducibility should be achievable when repeatedly approaching a focus position or an offset focus position. The aforementioned offset focus position is a distance between a reference position, for example a transition from an immersion medium to a coverslip covering the sample, and a depth position in the sample to be recorded. A further secondary requirement is that autofocusing should be as robust as possible vis-à-vis different samples, objectives, embedding media, etc. Furthermore, a high degree of robustness should also be achieved if the field of view contains no structures or only very weak structures of the samples to be imaged. By contrast, software-based autofocusing would only be possible in the case of a visible contrast in samples to be imaged. However, the measures required for hardware-based autofocusing should not be visible in the images to be output. The aforementioned secondary requirements further limit the possible solution space as well. A method known from the prior art for hardware-based autofocusing in microscopy is called definite focus and described by way of example in DE 10 2006 027 836 A1. According to this method, a grid is projected onto and reflected by a coverslip of a microscopic sample, and then detected by an oblique camera. A location of the sharpest image of the grid represents the focus position. Beam paths for illumination and detection of the grid are output coupled laterally from the regular beam path of the microscope. Illumination is implemented using electromagnetic radiation in the near infrared range. Internal offset focusing is implemented in an embodiment; in this case, an additional displaceable optical unit is used in order to be able to vary the focus position of the image on the camera. A further method known from the prior art for hardware-based autofocusing in microscopy is referred to as point triangulation autofocus and is described by way of example in DE 10 2017 218 449 B3. According to this method, a point is projected off-axis onto a coverslip of a sample, where it is reflected and then detected by a camera. An intensity centroid of the image of the point represents the focus position. Beam paths for excitation and detection of the point are output coupled laterally from the regular beam path of the microscope. Excitation is implemented using electromagnetic radiation in the near infrared range. There is internal offset focusing in one embodiment. Two points are imaged and detected in another embodiment. A further method known from the prior art for hardware-based autofocusing in microscopy is referred to as crescent triangulation autofocus and is described by way of example in JP 2004004634 A and US 2003/0184856 A1. According to this method, light is projected via a half-pupil onto a coverslip covering the sample, where it is reflected; it then passes another half-pupil in order to finally be detected by a camera. An intensity centroid of the half-pupil image represents the focus position. Beam paths for excitation and detection are output coupled laterally from the regular beam path of the microscope. Excitation