US-12618750-B2 - Method for generating a series of ultra-thin sections using an ultramicrotome, method for three-dimensional reconstruction of a microscopic sample, ultramicrotome system and computer program

US12618750B2US 12618750 B2US12618750 B2US 12618750B2US-12618750-B2

Abstract

A method for generating a series of ultra-thin sections of a microscopic sample is provided. The method includes detaching the sections from the sample using an ultramicrotome. The sections detached from the sample are made to float on a liquid surface and thereafter are transferred onto a solid carrier element. The method also includes determining, for at least one of the sections detached from the sample, a position and an orientation on the solid carrier element by monitoring the placement of the sections onto the solid carrier element using a monitoring system comprising a camera, and obtaining monitoring data.

Inventors

Robert Kirmse
Robert Lange
Peer Oliver Kellermann
Robert Ranner
Mario NOESSING

Assignees

LEICA MIKROSYSTEME GMBH

Dates

Publication Date: 20260505
Application Date: 20210617
Priority Date: 20200617

Claims (13)

1 . A method for three-dimensional reconstruction of a microscopic sample, the method comprising: detaching a series of ultra-thin sections from the sample using an ultramicrotome continuously, wherein the series of sections detached from the sample are made to float on a liquid surface of a water bath, and thereafter collecting and transferring the series of sections onto a solid carrier element, wherein the series of sections are being stretched caused by the floating to counteract deformations caused by the detaching; determining, for at least one of the each respective section of a plurality of consecutive sections detached from the sample, a position and an orientation on the solid carrier element by: acquiring moving-image data, using a camera of a monitoring system, as the plurality of consecutive sections are being transferred onto the solid carrier element, and monitoring the placements of the plurality of consecutive sections onto the solid carrier element using the monitoring system by tracking the plurality of consecutive sections in the moving-image data, so that the plurality of consecutive sections is associated with a sequence in which the plurality of sections is detached from the microscopic sample and hence with positions in the microscopic sample; obtaining monitoring data, wherein the monitoring data comprises for at least one of the sections detached from the sample for which the position and the orientation on the solid carrier element is determined, a position indicator and an orientation indicator relative to a reference position or orientation; microscopically investigating the series of sections of the microscopic sample using a microscopic device; and acquiring section image data, wherein the section image data are assembled into a volume image, and wherein the assembling the section image data into the volume image is performed based on the monitoring data obtained using the monitoring system or based on data derived from the monitoring data.
2 . The method according to claim 1 , comprising: dissecting, after placing the sections detached from the sample onto the solid carrier element, the solid carrier element into several carrier element parts; placing the carrier element parts onto a transfer device; and determining, for at least for one of the carrier element parts generated by dissecting the carrier element, a position and/or an orientation on the transfer device by monitoring the placement of the carrier element parts onto the transfer device using the same monitoring system or a further monitoring system.
3 . The method according to claim 1 , wherein the detachment of the series of sections from the sample encompasses the generation of section ribbons in which the sections adhere to each other, and in which the sections are placed at least in part in the form of such section ribbons onto the solid carrier element.
4 . The method according to claim 1 , wherein the detachment of the series of sections from the sample encompasses the generation of individual sections not adhering to each other, and in which the sections are placed at least in part in the form of such individual sections onto the solid carrier element.
5 . The method according to claim 1 , wherein the solid carrier element is provided to comprise machine-readable identifiers for target positions and/or reference positions of the sections to be placed onto the solid carrier element.
6 . The method according to claim 1 , wherein the section image data are acquired and/or assembled into the volume image by using an image acquisition system and/or an image analysis system, the monitoring data or the data derived from the monitoring data being transferred from the monitoring system to the image acquisition system and/or the image analysis system.
7 . The method according to claim 1 , further comprising: adjusting parameters of the microscopic device based on the monitoring data obtained using the monitoring system or the data derived from the monitoring data.
8 . The method according to claim 1 , comprising: dissecting, after placing the sections detached from the sample onto the solid carrier element, the solid carrier element into several carrier element parts; placing the carrier element parts onto a transfer device; and determining, for at least for one of the carrier element parts generated by dissecting the carrier element, an orientation on the transfer device by monitoring the placement of the carrier element parts onto the transfer device using the same monitoring system or a further monitoring system.
9 . The method according to claim 1 , wherein the solid carrier element is provided to comprise machine-readable identifiers for target positions of the sections to be placed onto the solid carrier element.
10 . The method according to claim 1 , wherein the section image data are acquired and/or assembled into the volume image by using an image analysis system, the monitoring data or the data derived from the monitoring data being transferred from the monitoring system to the image analysis system.
11 . A system for three-dimensional reconstruction of a microscopic sample, the system comprising: an ultramicrotome system for generating a series of ultra-thin sections of a microscopic sample, the ultramicrotome system being configured to detach the series of ultra-thin sections from the sample, to cause the series of sections to float on a liquid surface of a water bath, and to thereafter collect and transfer the series of sections onto a solid carrier element, wherein the series of sections are being stretched caused by the floating to counteract deformations caused by the detaching, the ultramicrotome system comprising: a monitoring system comprising a camera, the monitoring system being configured to: determine for at least one of the each respective section of a plurality of consecutive sections detached from the sample, a position and an orientation on the solid carrier element by: acquiring moving-image data using the camera as the plurality of consecutive sections are being transferred onto the solid carrier element, and monitoring placements of the plurality of consecutive sections onto the solid carrier element by tracking the plurality of consecutive sections in the moving-image data, so that the plurality of consecutive sections is associated with a sequence in which the plurality of sections is detached from the microscopic sample and hence with positions in the microscopic sample; and obtain monitoring data, wherein the monitoring data comprise or are used in generating a dataset comprising, for at least one of the sections detached from the sample for which the position and the orientation on the solid carrier element is determined, a position indicator and an orientation indicator relative to a reference position or orientation; and a microscopic device and an image analysis system comprising one or more processors configured to: microscopically investigate the series of sections of the microscopic sample, and acquire section image data, wherein the section image data is assembled into a volume image, and wherein the assembling the section image data into the volume image is performed based on the monitoring data obtained using the monitoring system or based on data derived from the monitoring data.
12 . The system according to claim 11 , comprising a feeding system configured to: provide the solid carrier element in the form of a ribbon-like structure; and position the solid carrier element for placing the sections detached from the sample thereon.
13 . A non-transitory computer-readable medium storing computer-executable program code for performing the method according to claim 1 when the computer program is executed by a processor.

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/EP2021/066400, filed on Jun. 17, 2021, and claims benefit to European Patent Application No. EP 20180467.1, filed on Jun. 17, 2020. The International Application was published in English on Dec. 23, 2021 as WO 2021/255163 A1 under PCT Article 21(2). FIELD The present invention relates to a method for generating a series of ultra-thin sections of a microscopic sample using an ultramicrotome, to a method for three-dimensional reconstruction of a microscopic sample from a series of sections, to an ultramicrotome system, and to a computer program. BACKGROUND Investigations of series of sections, in particular by electron microscopy, and reconstruction of three-dimensional sample information from such series, are very important, in particular in neuroscience but also in other fields of biology and medicine. Corresponding methods encompass, inter alia, “serial section scanning electron microscopy” (ssSEM, S3EM) and “serial section transmission electron microscopy” (ssTEM). In contrast to so-called thin sections generated using a regular microtome for light microscopy, having a typical thickness of about 5 μm, the section thicknesses in ultramicrotomy are generally below 1 μm or below 500 nm and for example between 10 and 200 nm or 25 and 100 nm, to enable them for use with different types of electron microscopy. In ultramicrotomy, glass or diamond blades are typically used. The ultra-thin sections are collected in a water trough and can be collected from there, as described below. Particularly as a result of the low sample thicknesses and the particular design of the equipment used, ultramicrotomy substantially differs from other methods of generating sample sections such the one disclosed in WO 2018/094290 A1, for example, where a sectioning process involves embedding of tissue in a support material such as a wax, resin, ice, or gel, and then slicing it using a microtome or vibrating blade microtome to a thickness in the order of micrometers to hundreds of micrometers, and where an attractive force pulls a tissue slice into contact with a support substrate or pallet as the tissue slice is being sectioned from the sample block in a water bath. As described, for example, in Horstmann, H. et al., Serial Section Scanning Electron Microscopy (S3EM) on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues, PLoS ONE 7(4), 2012, e35172, by means of ssSEM it is possible to provide a high-resolution, three-dimensional (3D) depiction of cellular ultrastructure. In contrast to ssTEM (explained below), which permits an investigation of restricted sub-cellular volumes but by no means a complete ultrastructural reconstruction of large volumes, entire cells, or entire tissues, the latter is possible using ssSEM. In ssSEM, serial sectioning of tissues is combined with scanning electron microscopy (SEM), in particular using a conductive wafer as a carrier. In ssSEM, section ribbons having hundreds of sections with a thickness of, for example, 35 nm may be generated, and are imaged on the wafer with a lateral pixel resolution of, for example 3.7 nm. Back-scattered electrons can be recorded using the “in-lens” detector of the SEM. The images resulting from such a method are comparable in quality to those of a conventional TEM. The essential advantage of ssSEM is that comparatively large structures, for example in the range of tens to hundreds of cubic micrometers, can be reconstructed with it. The more conventional ssTEM method is described, for example, in Harris, K. M. et al., Uniform Serial Sectioning for Transmission Electron Microscopy, J. Neurosci. 26(47), 2006, 12101-12103. ssTEM can also be superior to other methods for reconstructing three-dimensional sample information, such as confocal microscopy, in particular because of the high resolution. A sample for ssSEM and ssTEM is prepared for processing in known fashion and embedded, for example, in agarose or in suitable plastics. Section ribbons, in which the individual sections adhere to one another, are produced from the embedded sample using an ultramicrotome, by setting a suitable advance rate. Corresponding section ribbons float in a liquid bath and are removed using suitable transfer devices (“slot grids” or, in the case of ssSEM, also wafers) for further investigation. It is also possible not to allow the resulting section ribbons to float on a liquid bath but instead to transfer them directly onto a suitable carrier, for example a wafer. The location of an individual section in the specimen being investigated corresponds to its location in a section ribbon that has been generated, and vice versa. It may therefore be important, in corresponding methods, to generate section ribbons that are as long and uninterrupted as possible, thereby allowing the location of the individual sections in the sample as