Methods for generating a series of sections, methods for the three-dimensional reconstruction of a microscopic sample and microtome system

Abstract

Method (400) for producing a series of sections (11, AH) of a microscopic sample (10), wherein the sections (11, AH) are separated from the sample (10) as semi- or ultrathin sections by means of a blade (21) of an ultramicrotome (110), collected and applied to a transfer device (24), wherein - a sequence in which the sections (11, AH) are separated from the sample (10), and each of the sections (11, AH) is numbered according to the sequence of separation when separated from the sample (10), - the separation of the sections (11, AH) from the sample (10) and / or the collection of the sections (11, AH) and / or the application of the sections (11, AH) to the transfer device (24) is monitored by means of one or more observation cameras (30) while receiving moving image data, - the cuts (11, AH) in the moving image data can be tracked while maintaining their numbering, - Positions of the cuts (11, AH) on the transfer device (24) are related to the sequence of their separation from the sample (10) based on the tracking in the moving image data, and - an assignment of the positions of the sections (11, AH) on the transfer device (24) to the sequence of their separation from the sample (10) is carried out based on their numbering.

Description

[0001] The present invention relates to a method for producing a series of sections of a microscopic sample, a method for the three-dimensional reconstruction of a microscopic sample from a series of sections and a microtome system having the features of the independent patent claims. State of the art

[0002] Particularly in neuroscience, but also in other areas of biology and medicine, the examination of serial sections, especially by means of electron microscopy, and the reconstruction of three-dimensional sample information from such serial sections are of great importance.

[0003] From DE 10 2013 106 023 A1 a system and a method for producing fine tissue objects is known, wherein a slide and / or a fine tissue object applied to such a slide is automatically and unambiguously assigned to an object container and / or a tissue object removed from such an object container.

[0004] Corresponding methods include the so-called “Serial Section Scanning Electron Microscopy” (ssSEM, S 3EM) and the so-called "Serial Section Transmission Electron Microscopy" (ssTEM), whereby the present invention can be used in particular in conjunction with ssSEM. However, it is understood that the principles explained below can also apply in particular to other methods of a comparable nature. In particular, the present invention can in principle be used in conjunction with light microscopy instead of electron microscopy, although the following explanations focus on electron microscopy.

[0005] Using ssSEM, as described for example in Horstmann, H. et al., Serial Section Scanning Electron Microscopy (S 3 EM) on Silicon Wafers for Ultra-Structural Volume

[0006] Imaging of Cells and Tissues. PLoS ONE 7(4), 2012, e35172, describes how a high-resolution, three-dimensional (3D) representation of cellular ultrastructure can be obtained. In contrast to the ssTEM described below, which allows the investigation of limited subcellular volumes but hardly allows for complete ultrastructural reconstructions of large volumes, entire cells, or entire tissues, this is possible using ssSEM.

[0007] In ssSEM, serial sectioning of tissue is combined with scanning electron microscopy (SEM), particularly using a conductive wafer as a support. With ssSEM, section bands with hundreds of sections with a thickness of, for example, 35 nm can be created and imaged on the wafer with a lateral pixel resolution of, for example, 3.7 nm. Backscattered electrons can be recorded using the detector in the SEM objective (“in-lens detector”). The images resulting from this method are qualitatively comparable to those of a conventional TEM. The key advantage of ssSEM is that it allows the reconstruction of comparatively large structures, for example in the tens to three-digit cubic micrometer range.

[0008] The more conventional method, ssTEM, is described, for example, in Harris, KM 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, particularly due to its high resolution.

[0009] A sample is prepared for ssSEM and ssTEM in a conventional manner and embedded, for example, in agarose or suitable plastics. Using an ultramicrotome, section bands are created from the embedded sample by setting a suitable feed rate, with the individual sections adhering to one another. These section bands float in a liquid bath and are removed for further examination using suitable transfer devices (so-called slot grids or, in the case of ssSEM, wafers). It is also possible to transfer the resulting section bands directly to a suitable support, such as a wafer, rather than allowing them to float on a liquid bath.

[0010] The position of an individual section in the object under investigation corresponds to its position in a generated section band and vice versa. Therefore, in corresponding procedures it is of great importance to create section bands that are as uninterrupted and long as possible in order to be able to determine the position of the individual sections in the overall sample. As stated in the cited specialist literature, however, this cannot always be guaranteed, for example with certain embedding materials. For long section bands, correspondingly long liquid baths or recording devices are required, and handling the sections becomes more difficult. The targeted creation of multiple section bands also typically proves to be problematic in practice and requires skill and extensive practice.

[0011] One way to create multiple section bands using a liquid bath is to separate the currently adhering section band from the blade after a desired number of cuts using a manipulation tool (typically a scissor) and direct it to an area of ​​the liquid bath where it will not hinder the cutting of the new band. Once a sufficient number of section bands have been created in this way, the inserted transfer device is slowly lifted out of the liquid. The liquid drains away and the sections adhere to the transfer device. Typically, up to approximately 200 sections can be created in this way. If no liquid bath is used, the explanations apply accordingly.

[0012] As mentioned, one disadvantage of this method is that the sections of a section band often do not adhere reliably to one another. For example, sections of the band may drift apart on the liquid surface, making it impossible to subsequently assign them to the cutting sequence. Bending of the corresponding section bands is also frequently observed, causing them to collide with edge structures or other bands. This frequently leads to tearing and, in some cases, the resulting fragments drifting apart. If section bands or their fragments collide with the liquid boundary when using a liquid bath, they cannot be deposited on the transfer device, or cannot be deposited reliably, but instead continue to adhere preferentially to the liquid boundary. This can result in lost sections.A further disadvantage is that the transfer device can only be filled to a low level using the method described. This is typically 10% at most, so frequent transfers are necessary in such processes. Manipulation with the manipulation tool is also risky, as the tapes and cuts can be damaged. In particular, this can lead to tearing and / or holes or creases.

[0013] An alternative is to create parallel section bands, each attached to the blade. Up to 300 sections can be created in this way. In this context, however, it is necessary to position the sections of interest at a certain distance from the blade. This is achieved by using so-called "release" sections, which only serve the purpose of extending the section bands and thus pushing the sections of interest into the area of ​​the transfer device, but which consume sample material. The release sections themselves are lost for the actual examination. The interposed release sections prevent continuous section generation over larger sample areas, since the series of sections of interest are repeatedly interrupted by the release sections required for positioning and then missing for the subsequent examination.

[0014] It is also known to create individual sections instead of using cutting belts and to fish them out of a liquid bath one after the other using a conveyor belt before they can drift away. The conveyor belt is virtually endless, and the sequence of sections on the belt corresponds to the cutting sequence. However, this process entails a high level of manual effort. The conveyor belt must typically be cut by hand and then fixed to large silicon wafers for transfer to the microscope. This inevitably results in a low fill level of the silicon wafers with sections, resulting in high material consumption. Setting up the equipment is time-consuming, the corresponding equipment is expensive, and the conveyor belt material can be difficult to obtain.

[0015] Against this background, the present invention has the object of providing improved possibilities for creating series of sections that are suitable for a three-dimensional reconstruction of a corresponding sample. Disclosure of the invention

[0016] Against this background, the present invention proposes a method for generating a series of sections of a microscopic sample, a method for the three-dimensional reconstruction of a microscopic sample, and a microtome system having the features of the independent patent claims. Advantageous embodiments are the subject of the dependent claims and the following description. Advantages of the invention

[0017] In the method proposed according to the invention for producing a series of sections of a microscopic sample, the sections are separated from the sample using a suitable blade, as is known in the art, collected and then applied to a transfer device. Corresponding methods have already been explained in detail at the outset. The present invention is used in connection with electron microscopy methods and ultramicrotomes. So-called semi-thin sections or ultra-thin sections are produced here. In general, the sections produced within the scope of the present invention can have any thickness that can be produced using corresponding devices. For example, the sections produced with a diamond knife using a liquid bath typically have thicknesses of approximately 10 nm to 300 nm. With the ssSEM mentioned at the outset, these section thicknesses and in particular section thicknesses of approximately 10 to 100 nm or of approximately10 nm to 50 nm are used. Therefore, whenever reference is made to a "section" below, this includes microscopic sections of all typical section thicknesses for the aforementioned purposes.

[0018] The present invention particularly relates to the generation of sections for methods in which the sections are captured microscopically and three-dimensional data (volumetric image data) is reconstructed from the correspondingly acquired sections. As mentioned, this task requires, in particular, the generation of uninterrupted series of sections and the assignment of the respective sections to the order in which they were separated from the sample and thus to their position in the volumetric image data. The present invention enables this by capturing moving image data (video data) and tracking the generated sections in the moving image data. In this way, despite drifting movements, the disintegration of section bands, and the like, a reliable assignment of the individual sections with regard to their position in the entire object or in a corresponding volumetric image can always be achieved.It is known that consecutive cuts are often very similar and therefore it is difficult to assign them to another cut.

[0019] In principle, various devices for collecting the generated sections can be used within the scope of the present invention. As mentioned, the use of liquid baths is particularly known in this context, whereby the sections are collected on a corresponding liquid surface. A typical liquid is (deionized) water. However, the sections can also be collected on a solid support or its surface. The following explanations refer only to the collection of the sections on a liquid surface for the sake of clarity, but apply equally to other possibilities.

[0020] Within the scope of the present invention, a sequence in which the sections are separated from the sample is recorded, and the separation of the sections from the sample and / or the collection of the sections and / or the application of the sections to the transfer device is monitored using one or more observation cameras while receiving moving image data. Preferably, all three of the aforementioned steps are monitored in the manner explained. The observation cameras used can be set up for different observation modalities, for example for observation in the visual spectrum or in the infrared. Within the scope of the present invention, an "observation camera" is understood to mean any camera that makes it possible, in particular, to capture a surface on which the sections are collected, across the entire surface.Preferably, however, a corresponding observation camera is also configured to monitor, for example, the blade of the microtome used, identifying each section as it is created and subsequently tracking it. If necessary, refocusing can be performed, or multiple observation cameras focused on different planes can be used.

[0021] Within the scope of the present invention, the sections are each tracked in the moving image data, and the positions of the sections on the transfer device are correlated with the order of their separation from the sample based on their tracking in the moving image data. In this way, the sections can be localized on the transfer device according to the order of their separation from the sample. As already mentioned, this enables the respective sections on the transfer device to be assigned to their position in the sectioned sample or to images in a volume image.

[0022] In the case of ssSEM, the transfer device can be, in particular, a silicon wafer used for examination in a scanning electron microscope, or any other transfer device designed to examine a corresponding sample in a microscopic examination system or to transfer it into such a system. In the case of ssTEM, a known grid can be used, in particular. On a corresponding transfer device, the sections are treated, in particular, using suitable contrasting methods, for example, by vapor deposition or infiltration with dyes or heavy metal salts. However, such contrasting can also have already been carried out before the sample is subjected to the method according to the invention.

[0023] Within the scope of the present invention, the entire transfer device with the individual sections can be examined microscopically and the respective sections can be assigned to a volume image to be generated based on their previous tracking and position determination.

[0024] Within the scope of the present invention, the separation of at least some of the sections from the sample can comprise the creation of section bands, as fundamentally explained above. The sections can then be collected at least partially in the form of these section bands, for example on a liquid surface. Because the sample sections are tracked in the generated moving image data, these section bands can also be assigned accordingly. In contrast to the known methods explained at the outset, it is not necessary to deposit the section bands in a defined order, for example on a transfer device. The section bands can also be created with variable section band lengths, so that the complex creation of the longest possible section bands is no longer necessary and the associated problems can be avoided.In particular, this method prevents bending and breakage of excessively long section strips. Disintegration of section strips, which occurs more or less frequently depending on the sample, is also no longer a problem due to section tracking.

[0025] Within the scope of the present invention, a significantly denser packing of corresponding cuts on the transfer device can be achieved than in the prior art. This can be achieved, for example, by adjusting the number of cuts in a corresponding cut band or in several corresponding cut bands in order to achieve the densest possible cut band cloud or cut band packing in a region of the transfer device and, for example, previously on a liquid surface.

[0026] In other words, within the scope of the present invention, the cutting bands can be generated at least partially with a predetermined number of cuts or with a number of cuts within a predetermined range of numerical values, wherein the number of cuts in the cutting bands is predetermined in particular based on a target density of the cuts, for example on a liquid surface. In this context, the cutting process can be observed, for example, continuously or at predetermined time intervals, and the cutting position can be changed whenever it is detected that the density of the cuts exceeds a favorable value or a distance between the cuts falls below a favorable value, for example when it is visually observed that the cuts are in danger of abutting one another or overlapping one another. In this way, the cuts are brought to different positions in groups.A corresponding target density can also be specified, for example, in the form of a numerical value in an automated process, whereby the numerical value can be derived from empirical observations or theoretical data. Whenever the specified numerical value is exceeded, the cutting position can be changed.

[0027] A section band can be interrupted, for example, by setting a feed rate of zero in a corresponding microtome. If subsequent sections are made at the same point on a blade, for example a diamond blade, a subsequently created section band will push the first one in front of it until the first one deviates sideways, is twisted, and is no longer in the path of the second one. This is especially true if a liquid surface is used to collect the sections. If a large number of relatively short section bands are generated in this context, a particularly dense packing can be achieved. The optimal number of sections in the respective section bands can be easily determined empirically using "empty" sample material.

[0028] However, it can also happen that the resulting section band moves a large number of sections or fragments of sections. If the resistance becomes too great, this can lead to undesirable folding of the sections in thin sections. It can also happen that the newly created section bands push the existing section bands on the fluid surface out of a desired area.

[0029] Therefore, within the scope of the present invention, a targeted change of the cutting position can also be carried out. This can be achieved, for example, by cutting at a different point on a blade that is fixedly related to the respective collection device, for example a liquid surface. A corresponding blade can, for example, have a width of 4 mm, whereby the sample width can be, for example, 0.1 mm. In this way, a multitude of different position changes can be carried out. The generated cutting cloud can thus be made even wider and the area of ​​a transfer device can be better utilized. One criterion for a corresponding change can be, for example, that the last cut caused the sections to cross a spatial boundary on a liquid surface.This boundary can be selected such that all sections can be captured by the transfer device and none of the sections touch the boundary. A change in section positions can also be observed, for example, due to excessive displacement resistance or if the sections are folded. In other words, within the scope of the present invention, the sections can be applied to the liquid surface in groups at different positions for collection.

[0030] Within the scope of the present invention, tracking the positions of the sections can include detecting a movement of the sections during their separation from the sample and / or during their collection, for example, on a liquid surface, and / or during their application to the transfer device. All movement processes that would conventionally lead to a loss of the assignment of the respective sections to their position in the sample can be detected in this way within the scope of the present invention. Within the scope of the present invention, for example, various tracking algorithms can be used, as are generally known from the prior art.The tracking algorithms can, for example, be based at least in part on recording a film and only shifting the sections between the recordings of two film frames so small that, when comparing the film frames, the assignment is unambiguous due to the overlap. In this embodiment, closely spaced image pairs are continually compared, so that the assignment of the respective sections to their position in the sample is maintained. On the other hand, individual features such as shapes, colors, or structures within the moving sections can also support the assignment for tracking.

[0031] In the present invention, each of the sections is numbered upon separation from the sample, the tracking of the positions of the sections is carried out while maintaining their numbering, and the assignment of the positions of the sections on the transfer device to the order of their separation from the sample is carried out on the basis of the numbering. In this way, a particularly simple assignment can be carried out. In particular, based on the tracking of the sections within the framework of the present invention, a location map of the sections can be created, wherein the sections in the location map are numbered according to the order of their separation from the sample. The location map thus describes the position of the sections on the transfer device according to the order of their separation and can be used for the aforementioned assignment. The numbering can take place in real time, i.e.Each cut can be assigned a number during or after its creation, and each cut can then be tracked using this number. Alternatively, the numbering can also be performed after the editing process is completed using the recorded video data. In this case, tracking is also possible using the respective number.

[0032] As mentioned, the sections within the scope of the present invention are used or created as semi- or ultra-thin sections, and a glass or diamond blade can be used as the blade. The blades can be part of corresponding blade units, which can also have a tray for holding a liquid used to form a liquid level on which the sections can be collected.

[0033] The present invention also relates to a method for the three-dimensional reconstruction of a microscopic sample, in which sections of the sample are examined microscopically to obtain cross-sectional image data, and in which the cross-sectional image data are combined to form a volume image. This method is characterized in that the sections of the sample are generated using a method as previously explained in the embodiments, and in that a position of the cross-sectional image data in the volume image is determined based on the tracking of the positions of the sections in the moving image data and on the basis of the numbering carried out according to the invention. For features and advantages of a corresponding method and its advantageous embodiments, express reference is made to the above explanations, since these also relate to the inventive method for three-dimensional sample reconstruction.

[0034] In particular, the microscopic examination may include a light microscopic or an electron microscopic examination.

[0035] Furthermore, the present invention relates to a microtome system with an ultramicrotome and a data acquisition and evaluation unit and with one or more observation cameras, wherein the microscope is configured to produce a series of sections of a microscopic sample, and wherein the production of the series of sections comprises separating the sections from the sample by means of a blade, collecting them and then applying them to a transfer device.The microtome system is characterized in that it is configured to record an order in which the sections are separated from the sample, and to number each of the sections as they are separated from the sample according to the order of separation, and further to monitor the separation of the sections from the sample and / or the collection of the sections and / or the application of the sections to the transfer device while receiving moving image data by means of the one or more observation cameras.It is further characterized in that it is configured to track positions of the cuts in the moving image data by means of the data acquisition and evaluation unit and to relate positions of the cuts on the transfer device to the order of separation from the sample on the basis of the tracking in the moving image data, as well as to assign the positions of the cuts on the transfer device to the order of their separation from the sample on the basis of their numbering.

[0036] As mentioned several times, the collection of sections can be carried out particularly on a liquid surface, but also on a solid surface.

[0037] The invention is explained in more detail below with reference to the accompanying drawings, which illustrate embodiments of the invention. Short description of the drawings Fig. 1 shows a microtome system according to an advantageous embodiment of the invention in a highly simplified side view. Fig. 2 shows a blade unit of a microtome system usable in an advantageous embodiment of the invention in a highly simplified top view. The Fig. 3A to 3F illustrate a cut generation according to an advantageous embodiment of the invention in a highly simplified plan view. Fig. 4 illustrates a method according to an advantageous embodiment of the invention in the form of a schematic flow chart.

[0038] In the figures, structurally or functionally corresponding elements are indicated with identical reference symbols and are not explained repeatedly for the sake of clarity. Detailed description of the drawings

[0039] Fig. Figure 1 shows a microtome system according to one embodiment of the invention in a highly simplified side view. The microtome system is designated overall by 100.

[0040] In the example shown, the microtome system 100 comprises a microtome 110, which can be designed in particular as an ultramicrotome. Furthermore, a computing and evaluation unit 120 is provided, which is connected to the microtome 110 via a wired or wireless communication link 130. In contrast to the illustration of the Fig. 1, the computing and evaluation unit 120 can also be accommodated in the microtome 110 or a housing thereof or in a PC system or a workstation.

[0041] The microtome 110 has a sample holder 114, which can be advanced in the direction of an arrow 112 by means of a feed unit 111 to produce sections, and can also be moved up and down in the direction of an arrow 113, to which a sample 10 can be attached. During the up and down movement of the sample holder 114, the sample 10 is guided past a blade 21, in particular a diamond blade, which is attached to a blade unit 20, shown greatly enlarged and in a lateral sectional view, whereby a thin section is produced as a result of the feed. As explained in more detail with reference to the following figures, the sections produced in this way float in a liquid tray 22 of the blade unit 20 on a suitable liquid 23 or its liquid surface 25. The liquid surface 25 is flush with an upper edge of the liquid tray 22, so that the blade 21 is always wetted with liquid.

[0042] It should be emphasized again that, within the scope of the present invention, sections can alternatively be collected on a solid surface instead of the liquid surface 25. In this case, no liquid tray 22 is used, but rather a differently designed collection device. The following explanations relate to such a configuration in an analogous manner.

[0043] An observation microscope 115 is provided for monitoring the sectioning process. Instead of an observation microscope 115 or in addition thereto, a camera with a monitor can also be provided. The observation microscope 115 is attached to a support arm 116 of the microtome 110. The observation microscope 115 has one or more control elements 117 for adjustment. The microtome 110 has several control elements 118 for setting sectioning parameters. One or more observation cameras 30 can be installed on the observation microscope 115 or at one or more other suitable positions, by means of which the separation of the sections from the sample 10 and / or their transfer to the liquid surface 25 of the liquid 23 and / or their transfer to a transfer device not shown here can be monitored, as explained in detail above and below.For this purpose, the observation cameras 30 can be connected in particular to the computing and evaluation unit 120.

[0044] The Microtome 110 can Fig. 1, and any other devices, such as lighting devices, temperature control devices and the like.

[0045] In Fig. 2 is the blade unit 20, which in Fig. 1 is shown in a schematic side sectional view, is shown in a highly simplified plan view of the surface 25 of the liquid 23, which is parallel to the plane of the paper. In addition, a part of the sample holder 114 with the preferably frustoconically shaped sample 10 is illustrated. As mentioned, the blade 21 of a corresponding blade unit 20 can, for example, have a width of 4 mm, wherein the sample width can be, for example, 0.1 mm. The illustration in Fig. 2 and the following figures are therefore not to scale and the sections are shown greatly enlarged for clarity.

[0046] Through the aforementioned feed and up-and-down movement of the sample holder 114, sections 11 can be cut from the sample 10 by means of the blade 21 and applied to the liquid surface 25 of the liquid 23. Depending on the cutting parameters and materials used, the sections 11 are created in the form of individual cuts or as cutting strips 12 of the same or different lengths and, due to surface tension, lie freely on the liquid 23 without immersing it after being suitably detached from the blade 21.

[0047] After producing a sufficient number of cuts 11 or cutting bands 12, during which they can also be aligned and / or shifted, they can be transferred to a transfer device arranged below the cuts 11 or cutting bands 12, illustrated here by dashed lines and designated 24, by lowering the liquid surface 25 of the liquid 23. Any other relative movements between the cuts 11 or cutting bands 12 or the liquid surface 25 and the transfer device 24 are also possible in principle. Regarding the disadvantages of prior art methods, reference is made to the above explanations.

[0048] The Fig. 3A to 3F illustrate a cut generation in an advantageous embodiment of the invention in a highly simplified plan view, wherein the elements shown correspond to the already Fig. 2 explained elements. The Fig. 3A to 3F show snapshots during the creation of several sections 11 and section bands 12 in the same view as Fig. 2. The sections are designated here with capital letters to better understand the sequence of their creation. A representation of the transfer device 24, which may, however, already be present or arranged accordingly in corresponding steps, and a separate designation of the liquid surface were omitted in the Fig. 3A to 3E are omitted.

[0049] In Fig. 3A, a first thin section A is created, which here still adheres to the blade 21. Several up and down movements of the sample holder 114 later, by creating sections BD, which are connected to each other and to the thin section A, a section band 12 adhering to the blade 21 was created, as in Fig. 3B illustrates.

[0050] The cutting band 12 can be extended in any way and then detached from the blade 21, for example, by using a feed movement of zero or by means of a suitable manipulation tool. The cutting band 12 with the cuts A to D is thereby transferred to the liquid surface of the liquid 23 and can drift away from the blade 21, as in Fig. 3C illustrates.

[0051] Within the scope of the present invention, the sequence of the separation of the sections 11 or AD as part of the section band 12 from the sample 10 and / or their transfer to the liquid surface of the liquid 13 and / or their transfer to the transfer device 24 while receiving moving image data, for example by means of the Fig. 1, and the cuts 11 and AD are tracked in the moving image data in order to be able to locate a position of the cuts 11 and AD according to the order of their generation on the transfer device. In this way, for example, the drift of the cut tape 12 can be tracked.

[0052] According to Fig. 3C, another cut E is also created, which is also extended to a cutting band or, as in Fig. 3D, can be intentionally or unintentionally detached individually from the blade 21 and transferred to the liquid surface of the liquid 23. Here, too, a corresponding drift movement results, whereby in the context of the present invention, the cut F is now also additionally tracked. As in Fig. As further illustrated in Figure 3D, the cutting band 12 with cuts AD has now also drifted further. These cuts AD are also further tracked. Additionally, another cutting band 12' with cuts FH is created, which here still adheres to the blade 21 and is, for example, further extended.

[0053] According to Fig. In Figure 3E, thin section D has separated from section band 12 for unimportant reasons, and sections AC and D have drifted further. However, sections AC and D can be traced individually as previously explained. The same applies here to section F, which has also drifted further, and to the newly formed section band 12', which has been extended here to include further, not separately labeled sections.

[0054] According to Fig. 3F, the cutting of sample 10 is complete. A large number of sections 11 or AH and / or section bands 12, 12', not all of which are provided with reference symbols, are now located on the liquid surface of the liquid 13. These can be deposited on the transfer device 24 in the manner explained. Due to the tracking in the moving image data, the creation sequence of the sections 11 or AH can be traced despite different drift movements.

[0055] It is understood that the representation of the Fig. 2 and 3A to 3F is greatly simplified and schematic. In particular, much longer cutting strips 12, 12' with a much larger number of cuts 11 and AH, respectively, and a much larger total number of cutting strips 12, 12' or cuts 11 and AH, respectively, can be produced. As mentioned, the cuts 11 and AH can typically also be significantly smaller, particularly with respect to the blade 21.

[0056] Fig. 4 illustrates a method according to an advantageous embodiment of the invention in the form of a schematic flow chart 400 with steps 401 to 406.

[0057] In step 401, the generation of a section series is initiated. This can be the case, for example, when a new sample 10 has been clamped into a sample holder 114 or has been cut to a region of interest. At the same time, in step 401 or thereafter, the generation of moving image data by one or more observation cameras 30 begins.

[0058] The method continues with a step 402, in which a section 11 is cut from the sample 10. This section 11 is identified in the continuously recorded moving image data in a step 403 and is subsequently monitored as a monitoring object in the computing and evaluation unit 120. This is illustrated in a step 404, in which a corresponding monitoring object is added to a list of monitoring objects.

[0059] The method is carried out, as illustrated by an arrow 410, until a sufficient number of cuts 11 have been produced. Further steps, for example, for separating individual cuts and cut bands 12, 12', may also be provided.

[0060] Once a sufficient number of slices 11 have been generated, they can be transferred to a transfer device 24 in a step 405 in the manner explained. Due to the continuous tracking of the slices 11, the slice sequence of any slices 11 that may have drifted in the meantime is known. This information can later be used in a 3D reconstruction step 406, as illustrated by an arrow 420.

Claims

[1] Method (400) for producing a series of sections (11, AH) of a microscopic sample (10), in which the sections (11, AH) are separated from the sample (10) as semi-thin or ultra-thin sections by means of a blade (21) of an ultramicrotome (110), collected and applied to a transfer device (24), wherein - a sequence in which the sections (11, AH) are separated from the sample (10) is recorded, and each of the sections (11, AH) is numbered according to the sequence of separation when separated from the sample (10), - the separation of the sections (11, AH) from the sample (10) and / or the collection of the sections (11, AH) and / or the application of the sections (11, AH) to the transfer device (24) is monitored by means of one or more observation cameras (30) while receiving moving image data, - the cuts (11, AH) are tracked in the moving image data while maintaining their numbering, - positions of the sections (11, AH) on the transfer device (24) are related to the order of their separation from the sample (10) on the basis of the tracking in the moving image data, and - an assignment of the positions of the sections (11, AH) on the transfer device (24) to the order of their separation from the sample (10) is carried out on the basis of their numbering. [2] Method (400) according to claim 1, wherein the sections (11, AH) are collected on a solid support or on a liquid surface (25). [3] Method (400) according to claim 1 or claim 2, wherein separating at least some of the sections (11, AH) from the sample (10) comprises producing section bands (12, 12') and wherein the sections (11, AH) are collected at least partially in the form of these section bands (12, 12'). [4] Method (400) according to claim 3, wherein the cutting bands (12, 12') are produced at least partially with a predetermined number of cuts or with a number of cuts in a predetermined area. [5] Method (400) according to claim 4, wherein the number of cuts of the cutting bands (12, 12') is predetermined on the basis of a target density of the cuts (11, AH) on the liquid surface (25). [6] Method (400) according to one of the preceding claims, in which the cuts (11, AH) are brought to different positions in groups for collection. [7] Method (400) according to one of the preceding claims, wherein the tracking of the positions of the cuts (11, AH) comprises detecting a movement of the cuts (11, AH) during separation from the sample (10) and / or during collection on the liquid surface (25) and / or during application to the transfer device (24). [8] Method (400) according to one of the preceding claims, wherein the tracking of the positions of the cuts (11, AH) is carried out using one or more tracking algorithms. [9] Method (400) according to one of the preceding claims, in which a location map of the sections (11, AH) is created on the basis of the tracking of the sections (11, AH), wherein the sections (11, AH) are numbered in the location map according to the order in which they were separated from the sample (10). [10] Method (400) according to one of the preceding claims, in which a glass or diamond blade is used as the blade (21). [11] Method for the three-dimensional reconstruction of a microscopic sample (10), in which sections (11, AH) of the sample (10) are examined microscopically to obtain sectional image data and in which the sectional image data are combined to form a volume image, characterized bythat the sections (11, AH) of the sample (10) are generated by means of a method (400) according to one of the preceding claims and that a position of the sectional image data in the volume image is determined on the basis of tracking the positions of the sections (11, AH) in the moving image data and on the basis of their numbering. [12] A method according to claim 11, wherein the microscopic examination comprises a light microscopic or electron microscopic examination. [13] Microtome system (100) with an ultramicrotome (110), a data acquisition and evaluation unit (120) and with one or more observation cameras (30), wherein the ultramicrotome (110) is designed to produce a series of sections (11, AH) of a microscopic sample (10), wherein the production of the series of sections (11, AH) comprises separating the sections (11, AH) from the sample (10) by means of a blade (21), collecting them and applying them to a transfer device (24), wherein the microtome system (100) is designed to - to record a sequence in which the sections (11, AH) are separated from the sample (10) and to number each of the sections (11, AH) as they are separated from the sample (10) according to the sequence of separation, - further monitoring the separation of the sections (11, AH) from the sample (10) and / or the collection of the sections (11, AH) and / or the application of the sections (11, AH) to the transfer device (24) while receiving moving image data by means of the one or more observation cameras (30), - to track the positions of the cuts (11, AH) in the moving image data by means of the data acquisition and evaluation unit (120), - to relate the positions of the sections (11, AH) on the transfer device (24) to the order of separation from the sample (10) on the basis of the tracking in the moving image data, and - to assign the positions of the sections (11, AH) on the transfer device (24) to the order of their separation from the sample (10) on the basis of their numbering.

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