US-12625359-B2 - Method for providing position information for retrieving a target position in a microscopic sample, method for examining and/or processing such a target position and means for implementing these methods

US12625359B2US 12625359 B2US12625359 B2US 12625359B2US-12625359-B2

Abstract

A method for providing position information for retrieving a target position in a microscopic sample includes providing a first representation of the sample at a first resolution including the target position; specifying a first target position identifier indicating the target position at the first resolution; acquiring an image stack comprising the target position indicated by the first target position identifier; providing a second representation at a second resolution higher than the first resolution based on the image stack; specifying a second target position identifier indicating the target position at the second resolution; specifying a plurality of reference position identifiers in the second representation indicating positions of optically detectable reference markers at the second resolution; and determining a set of geometric descriptors describing spatial relations between the second target position identifier and the plurality of reference position identifiers to provide the position information.

Inventors

Frank Sieckmann
Frank Hecht

Assignees

LEICA MICROSYSTEMS CMS GMBH

Dates

Publication Date: 20260512
Application Date: 20211102

Claims (17)

1 . A method for providing position information for retrieving a target position in a microscopic sample, the method comprising: a) providing a first digital representation of the sample or a part of the sample at a first resolution comprising the target position; b) specifying a first target position identifier in the first digital representation indicating the target position at the first resolution; c) acquiring an image stack in a region of the sample comprising the target position indicated by the first target position identifier, the image stack comprising a plurality of images; d) providing a second digital representation at a second resolution higher than the first resolution based on the image stack by performing a deconvolution between pixels of each respective image of the image stack and between pixels of different images of the image stack; e) specifying a second target position identifier in the second digital representation indicating the target position at the second resolution; f) specifying a plurality of reference position identifiers in the second digital representation indicating positions of optically detectable reference markers at the second resolution; and g) determining a set of geometric descriptors describing spatial relations between the second target position identifier and the plurality of reference position identifiers to provide the position information.
2 . The method according to claim 1 , wherein the target position is a first target position, wherein the method is adapted for providing position information for retrieving one or more further target positions by performing steps b) to g) for each of the one or more further target positions, and wherein the regions of the sample in which the image stacks are acquired for the first and each of the one or more further target positions are disjoint regions.
3 . The method according to claim 1 , wherein in step f) the plurality of reference position identifiers in a distance larger than a lower distance threshold to the target position and/or in a distance smaller than an upper distance threshold to the target position are specified in the second digital representation.
4 . The method according to claim 1 , wherein a light microscopic device is used for acquiring the image stack and wherein the second resolution is a digital resolution exceeding an optical resolution of the light microscopic device.
5 . The method according to claim 4 , wherein providing the second digital representation includes interpolating image pixels within and/or between individual images of the image stack to yield the second resolution.
6 . The method according to claim 1 , wherein providing the first digital representation comprises computationally merging a plurality of light microscopic images, the plurality of light microscopic images being acquired at least in part at different lateral positions and/or different focus positions.
7 . The method according to claim 1 , wherein providing the first digital representation comprises displaying at least a part of the first digital representation in a first display region of a graphical user interface rendered by a computing device on a display.
8 . The method according to claim 7 , wherein specifying the first position identifier comprises receiving and processing a first user input of a user of the computing device, the first user input indicating a position in the first display region of the graphical user interface.
9 . The method according to claim 7 , wherein providing the second digital representation comprises obtaining a three-dimensional representation based on the image stack and displaying one or a plurality of two-dimensional views of the three-dimensional representation in a second display region of the graphical user interface.
10 . The method according to claim 9 , wherein specifying the second target position identifier includes receiving and processing a second user input of a user of the computing device, the second user input indicating a position in the second display region of the graphical user interface.
11 . The method according to claim 1 , further comprising specifying coarse reference position identifiers at the first resolution in the first digital representation indicating coarse positions of visible reference markers in the microscopic sample.
12 . The method according to claim 1 , wherein the set of geometric descriptors is determined as a vector set or a directed graph, and/or wherein the position information is provided in a form of coordinates relative to a reference point determined based on or relating to the vector set or the directed graph.
13 . The method according to claim 1 , wherein providing the position information comprises modifying the geometric descriptors based on an estimate of a shrinkage of the sample in a subsequent process.
14 . The method according to claim 1 , further comprising providing the position information to a processing device for further processing of the sample.
15 . A method for examining and/or processing the target position in a microscopic sample using a processing apparatus, wherein the position information for the target position is provided by a method according to claim 1 , and wherein the target position is retrieved in the processing apparatus on the basis thereof.
16 . A microscopic examination arrangement comprising a microscopic device and a computing device, the microscopic examination arrangement being adapted to perform the method according to claim 1 .
17 . A non-transitory computer readable medium comprising instructions thereon that, when executed by one or more processors, facilitate performing the method according to claim 1 .

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2021/080374, filed on Nov. 2, 2021. The International Application was published in English on May 11, 2023 as WO 2023/078527 A1 under PCT Article 21(2). FIELD The present invention relates to a method for providing position information for retrieving a target position in a microscopic sample, a method for examining and/or processing a microscopic sample at such a target position, and means for implementing these methods in the form of an apparatus, an examination arrangement and a computer program. BACKGROUND As mentioned in C. Kizilyaprak et al., “Focused ion beam scanning electron microscopy in biology”, J. Microsc. 254(3), 109-114, focused ion beam scanning electron microscopy (FIB-SEM) is progressively used in biological research. A focused ion beam scanning electron microscopy instrument is a scanning electron microscope (SEM) with an attached gallium ion column in which beams of electrons and ions may be focused to coincident points. One application of focused ion beam scanning electron microscopy is the acquisition of three-dimensional tomography data wherein, with the ion beam, thin layers of the surface at a target region are repetitively removed and the remaining block-face is imaged with the electron beam in a likewise repetitive manner. A focused ion beam scanning electron microscopy instrument can also be used to cut open structures for getting access to internal structures or to prepare thin lamellas for imaging by (cryo-)transmission electron microscopy. SUMMARY In an embodiment, the present disclosure provides a method for providing position information for retrieving a target position in a microscopic sample. The method includes the steps of: a) providing a first digital representation of the sample or a part of the sample at a first resolution including the target position; b) specifying a first target position identifier in the first digital representation indicating the target position at the first resolution; c) acquiring an image stack in a region of the sample including the target position indicated by the first target position identifier; d) providing a second digital representation at a second resolution higher than the first resolution based on the image stack; e) specifying a second target position identifier in the second digital representation indicating the target position at the second resolution; f) specifying a plurality of reference position identifiers in the second digital representation indicating positions of optically detectable reference markers at the second resolution; and g) determining a set of geometric descriptors describing spatial relations between the second target position identifier and the plurality of reference position identifiers to provide the position information. BRIEF DESCRIPTION OF THE DRAWINGS Subject matter of the present disclosure will be described in even greater detail below based on the exemplary figures. All features described and/or illustrated herein can be used alone or combined in different combinations. The features and advantages of various embodiments will become apparent by reading the following detailed description with reference to the attached drawings, which illustrate the following: FIG. 1 illustrates a graphical user interface in a general view; FIGS. 2A to 2D illustrate the user interface of FIG. 1 in different method steps; FIG. 3 illustrates a geometrical descriptor; FIG. 4 illustrates categories in defining a geometrical descriptor; FIG. 5 illustrates a method in the form of a flow diagram; FIG. 6 illustrates a computerized microscope system; and FIGS. 7A and 7B illustrate a user interface of a computerized microscope system. DETAILED DESCRIPTION Embodiments of the present invention overcome the present disadvantages of techniques using focused ion beam scanning electron microscopy and other methods of examining and processing microscopic samples. In an embodiment, a method for providing position information for retrieving a target position in a microscopic sample is proposed. The method comprises the steps of (a) providing a first digital representation of the sample or a part thereof at a first resolution including the target position, (b) specifying a first target position identifier in the first digital representation indicating the target position at the first resolution, (c) acquiring an image stack in a region of the sample including the target position indicated by the first target position identifier, (d) providing a second digital representation at a second resolution higher than the first resolution on the basis of the image stack, (e) specifying a second target position identifier in the second digital representation indicating the target position at the second resolution, (f) specifying a plurality of reference position identifiers in the second digital deta