Out-of-order scanning and automatic sequencing method for continuous ultrathin sections of electron microscope sample

Abstract

The invention provides an electron microscope sample continuous ultra-thin section out-of-order scanning and automatic sorting method which comprises the following steps: S1, preliminarily cutting a sample tissue subjected to permeation and embedding into a sample tissue structure with a special-shaped side surface; s2, gradually slicing from the narrow end of the special-shaped side surface of the sample tissue to the other end to obtain a plurality of slice samples, and scanning out of order; and S3, obtaining the section surface side length of each section through an electron microscope, and sorting according to the section surface side lengths to obtain a sorting result of the section sequence of the sample tissues. According to the method, the sample tissue is set to be of a special-shaped structure, slicing is started from the narrow end of the special-shaped side face, size comparison is carried out according to the side length of the slice face of the slices, and therefore the multiple slices are automatically sequenced in sequence, the position of each slice in the original sample tissue is restored, and the sequenced slices can be conveniently and rapidly sequenced through the image processing technology. And reconstructing an original sample tissue image.

Description

Technical Field

[0001] The invention relates to the field of electron microscope samples, in particular to an automatic sorting method for random scanning of continuous ultra-thin sections of electron microscope samples. Background Art

[0002] Electron microscopy is an ultra-thin sample preparation technology for observation using an electron microscope. In the fields of biology, medicine, and materials science, in order to be able to observe cell structures, tissue morphology, or material microstructures in detail, it is usually necessary to make samples into very thin slices. The thickness of the slices is generally between a few nanometers and tens of nanometers, so that the electron beam can penetrate and produce a clear image. Currently, most biological tissues are imaged by electron microscopy using continuous sectioning and scanning electron microscopy imaging. Among them, continuous sectioning uses an ultrathin slicer to continuously cut the tissue sample into multiple slices, and then collects them and places them in an electron microscope for imaging. However, the collected slices are randomly distributed, making it difficult to determine the original position of each slice in the original tissue, and the subsequent image registration process takes a long time. Chinese patent CN114049252A discloses a scanning electron microscope three-dimensional image acquisition system and method for serial sections, which records a scanning electron microscope three-dimensional image acquisition method for serial sections, the method comprising acquiring serial sections of biological tissue samples, determining the region of interest of each section, and generating the three-dimensional imaging instruction and the registration instruction of the region of interest; generating the control instruction of the control electron microscope according to the three-dimensional imaging instruction; the scanning electron microscope acquires the image data corresponding to the section according to the control instruction; performs two-dimensional splicing of the image data according to the registration instruction to obtain the microscopic image; and performs registration of the spliced ​​microscopic images according to the registration instruction and combines them in the order of the sections to obtain the three-dimensional image of the biological tissue sample. Although the method sets the region of interest, its data processing is still relatively time-consuming, and the registration processing is directly performed on the disordered section images and then the original tissue image is restored. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples, so as to solve the problem that in the continuous sections of electron microscope samples, the sections are randomly distributed and the section sorting cannot be obtained, resulting in a long time-consuming section image reconstruction.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for automatically sorting the random scanning of continuous ultra-thin sections of electron microscope samples, the method comprising the following steps: S1. Preliminary cutting of the sample tissue after infiltration and embedding into a sample tissue structure with a special-shaped side surface; S2, slicing the sample tissue from the narrow end of the irregular side to the other end, obtaining multiple slice samples, and scanning them in random order; S3. Obtain the side length of each slice through an electron microscope, sort the slices according to the side lengths, and obtain the sorting result of the slice order of the sample tissue.

[0005] In a preferred embodiment, step S2 comprises the following steps: S21, selecting the irregular side surface as the cutting surface according to the side length of the sample tissue, and the narrow end of the irregular side surface is the starting end of the tissue section; S22, setting the thickness of the sample tissue slices, cutting sequentially from the starting end of the special-shaped side surface, and obtaining multiple slices of the same thickness through a slicer; S23. Multiple slices are randomly distributed, and each slice is scanned by an electron microscope to obtain a slice surface image.

[0006] In the preferred embodiment, step S3 comprises the following steps: S31, extracting the slice surface side length data of the slice according to the slice surface image of each slice; S32, selecting the corresponding slice side length data for comparison, inserting into the interval range for sequential sorting; S33, sequentially sorting and marking the slices in order to form a slice sequence of the original sample tissue.

[0007] In the preferred embodiment, step S32 also includes the following steps: S321, obtaining the corresponding slice surface side length data of two slices, and setting them as a first interval range in order of size; S322, obtaining the slice surface side length data corresponding to the third slice, and comparing the length with the first interval range to form a second interval range; S323, repeat the above steps to compare the corresponding slice surface side length data of other slices until the slice surface side length data of all slices are sorted in sequence within the interval range.

[0008] In the preferred solution, or step S32, the maximum side length data or the minimum side length data of each slice surface are compared and sorted in sequence.

[0009] In a preferred embodiment, the method further comprises step S4, calculating the slice surface area or the slice volume of the slice, sorting the slice order twice according to the size of the calculated result, and verifying the sorting results of the two slice orders.

[0010] In a preferred embodiment, if the sorting results of the two slice sequences are different, steps S3 and S4 are repeated until the sorting results of the two slice sequences are the same, thereby constructing a slice sequence of the original sample tissue.

[0011] The present invention provides a method for automatically sorting the random scanning of continuous ultra-thin sections of electron microscope samples, the method comprising the following steps: S1, preliminarily cutting the sample tissue after infiltration embedding, cutting it into a sample tissue structure with a special-shaped side; S2, slicing from the narrow end of the special-shaped side of the sample tissue to the other end, obtaining multiple slice samples, and scanning them in random order; S3, obtaining the side length of the slice surface of each slice through the electron microscope, sorting the size according to the side length of the slice surface, and obtaining the sorting result of the slice sequence of the sample tissue. The method sets the sample tissue as a special-shaped structure, starts cutting from the narrow end of the special-shaped side, and compares the size according to the side length of the slice surface of the slice, thereby automatically sorting the multiple slices in order to restore the position of each slice in the original sample tissue, so that the sorted slices can be reconstructed into the original sample tissue image through image processing technology later. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Figure 1 It is a flow chart of the present invention; Figure 2 It is a cross-sectional view of the tissue of the physical sample of the present invention; Figure 3 It is a side view of the physical sample organization of the present invention Figure 4 It is a schematic diagram of the sample organization of the present invention; Figure 5 is a schematic diagram of random sections of the sample tissue of the present invention; Figure 6 It is a schematic diagram of the automatic sorting slices of the sample tissue of the present invention. DETAILED DESCRIPTION

[0013] Embodiment 1: like Figures 1 to 6 As shown, a method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples, the method comprising the following steps: S1, obtaining samples for pre-fixation, cleaning, post-fixation, immersion, dehydration and infiltration embedding treatment, and performing preliminary cutting of the sample tissue after infiltration embedding to cut it into a sample tissue structure with irregular side surfaces; preferably, the sample tissue is initially cut into a multi-faceted trapezoidal body, and the irregular side surfaces are trapezoidal surfaces.

[0014] S2. Slice the sample tissue from the narrow end of the irregular side surface to the other end to obtain multiple slice samples, and scan them in random order; preferably, fix the sample tissue after preliminary cutting in the sample clamping groove of the slicer, and slice continuously from the narrow end of the trapezoidal surface to the long end in sequence to cut out multiple trapezoidal slices from small to large, and the slices are randomly distributed in the collection tank, and the slice surface of the random slices is photographed and scanned.

[0015] S3, obtain the side length of the slice surface of each slice by electron microscopy, sort the size according to the side length of the slice surface, and obtain the sorting result of the slice order of the sample tissue. Preferably, select the trapezoidal slice surface as the comparison data, compare the side length of the trapezoidal slice surface of each slice, that is, compare the side length corresponding to the trapezoidal slice surface, and sort the slices in order from small to large according to the comparison results. Through the above steps, the automatic sequential sorting of the slices in the continuous ultrathin slices of the electron microscope sample is realized, so as to restore each slice to the original sequence position in the sample tissue.

[0016] In a preferred embodiment, step S2 comprises the following steps: S21, selecting the irregular side surface as the cutting surface according to the side length of the sample tissue, and the narrow end of the irregular side surface is the starting end of the tissue section; S22, setting the thickness of the sample tissue slices, cutting sequentially from the starting end of the special-shaped side surface, and obtaining multiple slices of the same thickness through a slicer; S23, multiple slices are randomly distributed, and each slice is scanned by an electron microscope to obtain a slice surface image. Preferably, the trapezoidal surface of the sample tissue is selected as the cutting surface, the end close to the narrow side of the trapezoidal surface is the starting end, and the end close to the long side of the trapezoidal surface is the ending end; the sample tissue is cut into multiple slices of the same thickness from the narrow end to the long end of the trapezoidal surface by a slicer, and a collection tank is provided below the slicer, and the slices are randomly distributed in the collection tank, and the slice surface of each slice is photographed and scanned one by one by using an electron microscope.

[0017] In the preferred embodiment, step S3 comprises the following steps: S31, extracting the slice surface side length data of the slice according to the slice surface image of each slice; S32, selecting the corresponding slice side length data for comparison, inserting into the interval range for sequential sorting; S33, sequentially arranging and marking the slices in order to form a slice sequence of the original sample tissue. Preferably, the side length data corresponding to the slice surface is randomly selected as the comparison data, and after the size comparison of the side length data of the slice surface corresponding to all the slices is completed, they are sequentially set within the interval range, and the slices are assigned values ​​and marked in order to form a slice sequence.

[0018] In the preferred embodiment, step S32 also includes the following steps: S321, randomly obtain the corresponding slice surface side length data of two slices, and set them as a first interval range in order of size; S322, randomly obtain the slice surface side length data corresponding to the third slice, and compare the length with the first interval range to form a second interval range; S323, repeat the above steps to compare the corresponding slice surface side length data of other slices until the slice surface side length data of all slices are sorted in sequence within the interval range.

[0019] In the preferred solution, or step S32, the maximum side length data or the minimum side length data of each slice surface are compared and sorted in sequence.

[0020] In a preferred embodiment, the method further comprises step S4, calculating the slice surface area or the volume of the slice, sorting the slice order twice according to the size of the calculation result, and verifying the sorting results of the two slice orders. Preferably, the slice surface area of ​​each slice is calculated for size comparison, and the automatic order sorting of the slices is completed, and the consistency of the sorting results of the two slice orders is compared. Preferably, or the volume of each slice is calculated, and the slice order is sorted according to the volume size.

[0021] In the preferred embodiment, if the sorting results of the two slice sequences are different, steps S3 and S4 are repeated until the sorting results of the two slice sequences are the same, and the slice sequence of the original sample tissue is constructed. The two slice sortings are used to verify the accuracy of the sorting results. Embodiment 2: Further illustrate with reference to Example 1, Figure 2~3 The structure shown in the figure, the operation flow of the method is as follows: pre-fix, wash, post-fix, immerse, dehydrate and infiltrate embed the sample; clamp the sample tissue after infiltration embedding in a special clamp, perform preliminary cutting, select the cutting surface of the sample tissue, and position and fix the sample in the microtome clamping groove, with the top exposed about 2mm; place the diamond knife or the glass knife with a water tank in the clamping gap of the knife table and fix it tightly, so that the blade is at the same height as the ruler next to the knife table clamp, adjust the position of the knife edge, and set the slice window; adjust the height of the collection tank, set the slice thickness to 50~70nm, the diamond knife slice rate to 1mm / s or the glass knife slice rate to 2~5 mm / s; according to the adjusted position and set parameters, the slicer is started for automatic continuous slicing, and the obtained slices are collected in the collection slot of the diamond knife or glass knife; the edge of the grid is clamped with elbow precision tweezers and bent so that the side of the grid with the support film faces down and forms a 45° angle with the slice, and the slice is quickly and lightly touched to make the slice flatly attached to the support film of the grid, and the edge of the grid is touched with filter paper to absorb moisture; the grid slice is stained, and the stained grid slice is placed in the sample rod, and subsequent shooting and scanning are performed through the electron microscope, and the slice sorting result of the sample tissue is obtained through the electron microscope sample continuous ultra-thin slice random scanning automatic sorting method.

[0022] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limiting the present invention. The protection scope of the present invention shall be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples, characterized by: The method comprises the following steps: S1. Preliminary cutting of the sample tissue after infiltration and embedding into a sample tissue structure with a special-shaped side surface; S2, slicing the sample tissue from the narrow end of the irregular side to the other end, obtaining multiple slice samples, and scanning them in random order; S3. Obtain the side length of each slice through an electron microscope, sort the slices according to the side lengths, and obtain the sorting result of the slice order of the sample tissue.

2. According to claim 1, a method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples is characterized by: Step S2 includes the following steps: S21, selecting the irregular side surface as the cutting surface according to the side length of the sample tissue, and the narrow end of the irregular side surface is the starting end of the tissue section; S22, setting the thickness of the sample tissue slices, cutting sequentially from the starting end of the special-shaped side surface, and obtaining multiple slices of the same thickness through a slicer; S23. Multiple slices are randomly distributed, and each slice is scanned by an electron microscope to obtain a slice surface image.

3. According to claim 1, a method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples is characterized by: Step S3 includes the following steps: S31, extracting the slice surface side length data of the slice according to the slice surface image of each slice; S32, selecting the corresponding slice side length data for comparison, inserting into the interval range for sequential sorting; S33, sequentially sorting and marking the slices in order to form a slice sequence of the original sample tissue.

4. According to claim 3, a method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples is characterized by: Step S32 also includes the following steps: S321, obtaining the corresponding slice surface side length data of two slices, and setting them as a first interval range in order of size; S322, obtaining the slice surface side length data corresponding to the third slice, and comparing the length with the first interval range to form a second interval range; S323, repeat the above steps to compare the corresponding slice surface side length data of other slices until the slice surface side length data of all slices are sorted in sequence within the interval range.

5. According to claim 3, a method for automatically sorting random scanning of continuous ultra-thin sections of electron microscope samples is characterized by: Or step S32, comparing the maximum side length data or the minimum side length data of each slice surface and sorting them in sequence.

6. The method for automatically sorting random scanning of continuous ultrathin sections of electron microscope samples according to claim 1, characterized in that: The method further comprises step S4, calculating the slice surface area or the slice volume of the slice, sorting the slice sequence twice according to the size of the calculated result, and verifying the sorting results of the two slice sequences.

7. The method for automatically sorting random-sequence scanning of continuous ultrathin sections of electron microscope samples according to claim 6, characterized in that: If the sorting results of the two slice sequences are different, step S3 and step S4 are repeated until the sorting results of the two slice sequences are the same, and the slice sequence of the original sample tissue is constructed.

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