CN117783168A - Cross-scale comprehensive analysis method for internal structure of fossil sample - Google Patents

Abstract

The invention discloses a trans-scale comprehensive analysis method for an internal structure of a fossil sample, which comprises the following steps: scanning the fossil sample in a high-resolution scanning mode by using the 3D-XRM to obtain high-resolution 3D-XRM data, and determining an ROI (region of interest) in the fossil sample; manufacturing an embedding block with scale marks on the surface; scanning the embedded block in a low resolution mode by using 3D-XRM to obtain low resolution 3D-XRM data; aligning the low-resolution data with the high-resolution 3D-XRM data, and determining the relative position of the interested region inside the fossil sample and the scale on the surface of the embedding block; scribing and positioning are carried out on the surface of the embedded block, directional cutting is carried out on the embedded block, the thin sheet exposed with the ROI is tested by using the FIB-SEM, and three-dimensional reconstruction is carried out; the high-resolution 3D-XRM data is imported into VGstudio Max software, so that analysis results of the same fossil in a trans-scale-multi-mode by combining two technologies are obtained. The invention realizes quick, economical and effective positioning to the position of the region of interest in the fossil sample.

Description

Cross-scale comprehensive analysis method for internal structure of fossil sample

Technical Field

The invention relates to the technical field of fossil analysis and test, in particular to a trans-scale comprehensive analysis method for internal structures of fossil samples.

Background

Ancient biology reconstructs the evolutionary process of biocenosis in geological stages by studying the morphology and anatomy of fossil. Two-dimensional imaging studies of fossils are commonly performed using conventional optical and scanning electron microscopes. With the intensive research, ancient biologists need innovative methods to obtain information of three-dimensional structures and in-situ chemical components simultaneously from different types of fossil samples. Because these information can improve the accuracy of judging the phylogenetic position of fossils in living trees, revealing their evolutionary significance in ancient biology.

In recent years, X-ray microscopy tomography (3D-XRM) systems have become widely used to obtain information about the appearance and internal structure of a sample. The technology can realize the three-dimensional visualization of the shape and the internal structure of the target object without damage. However, 3D-XRM itself does not provide information on fossil chemical composition. On the other hand, a focused ion beam scanning electron microscope (FIB-SEM) is a technique capable of acquiring structural and compositional information from fossil at the same time. The in-situ structure can be analyzed for elemental composition and crystal structure orientation using FIB-SEM and its matched X-ray energy scattering spectroscopy (EDS) and electron back scattering diffraction analyzer (EBSD), and resolution is improved by nearly 100 times compared to 3D-XRM. But its field of view is very limited and it is not easy to locate a region of interest (which may be simply called ROI) inside the sample. Although both the above techniques have their advantages, each has limitations that if the two techniques can be used in combination to perform cross-scale (different resolution) -multi-modal (different data types) testing on the same fossil, and perform data combination, the capability of scientific researchers to gather fossil structure and component information will be greatly improved.

The combined application of the two technologies can be expected to bring help to the ancient biology challenges which are difficult to solve, namely, the combined application of the two technologies in the ancient biology field can be rapidly increased. However, the current research situation does not reflect the expected growth, nor does it realize a better acceleration of the development and application of trans-scale comprehensive analysis of the specific structures inside the fossil. The root cause behind this discrepancy is:

1) The paleontological fossils have large internal anisotropy and are difficult to expose to internal specific ROIs. Unlike rock (mudstone, shale, etc.), relatively homogeneous samples such as pharmaceuticals, or artificially prepared samples, a large number of cut surfaces can be exposed by random cutting of a large number of samples, and then screening and combination testing can be performed on these cut surfaces. The quantity of the ancient fossil samples is small, and sometimes only one or a few blocks of fossil samples are exposed by adopting a random cutting mode, so that the efficiency is low, the loss of the rare samples is increased, the rare samples are even cut, and the test requirement cannot be met at all.

2) The cost of the mainstream FIB-SEM equipment is up to millions to tens of millions of RMB, the higher-configured equipment can be provided with a high-efficiency femtosecond laser sample processor, so that the efficiency and the scale of sample processing can be enlarged, but the higher-configured equipment also needs to cost an additional millions of prices, and the application of the equipment in the national range is further hindered. In addition, when 3D-XRM and FIB-SEM are combined, the processing capability of the FIB-SEM is very limited, so that the preliminary processing of fossil samples is time-consuming, high in cost and low in efficiency for large-scale structures.

Disclosure of Invention

In view of the above, the present invention is directed to a method for cross-scale analysis of internal structures of fossil samples, which is used to solve at least one of the above problems in the prior art.

The aim of the invention is mainly realized by the following technical scheme:

a method for trans-scale integrated analysis of internal structures of fossil samples, comprising:

and (B) step (B): scanning the fossil sample in a high-resolution scanning mode by using the 3D-XRM to obtain high-resolution 3D-XRM data; three-dimensional reconstruction is carried out based on 3D-XRM data to obtain an internal structural image of the fossil sample, and an ROI (region of interest) in the fossil sample is determined;

step C: manufacturing a fossil sample into an embedding block with scale marks on the surface;

step D: using 3D-XRM to rapidly scan the embedded block in a low resolution mode to obtain low resolution 3D-XRM data; and aligning the low-resolution data obtained in the step with the high-resolution 3D-XRM data obtained in the step B in three-dimensional image processing software, and determining the relative position of the region of interest inside the fossil sample and the scale on the surface of the embedding block;

step E: marking and positioning on the surface of the embedded block based on the determined relative positions of the region of interest inside the fossil sample and the scales on the surface of the embedded block so as to determine a surface to be cut; directionally cutting the embedding block along the surface to be cut by using a cutting machine to obtain a sheet containing the ROI, and grinding one of the cutting surfaces to obtain a sheet exposing the ROI;

Step G: performing test analysis on the thin sheet exposed with the ROI by using the FIB-SEM, and importing the obtained FIB-SEM three-dimensional data into VGstudio Max software for three-dimensional reconstruction;

step H: and (3) introducing the high-resolution 3D-XRM data into VGstudio Max software containing the FIB-SEM three-dimensional data, and performing data processing and fine alignment to obtain a cross-scale multi-mode analysis result of the same fossil by combining the two technologies.

Further, between step E and step G, step F: the ROI is further exposed or polished.

Further, step B includes:

step B1: scanning a target fossil sample with a resolution of 0.5-3 μm by using a 3D-XRM, wherein the scanning time is 3-8 hours, obtaining high-resolution 3D-XRM data, and accurately positioning the ROI based on the high-resolution 3D-XRM data;

step B2: carrying out data processing and three-dimensional reconstruction on the high-resolution 3D-XRM data to obtain the volume data of the fossil sample;

step B3: based on the volume data, a three-dimensional visual expression result of the fossil sample is obtained.

Further, the step C comprises the following steps:

step C1: placing the fossil sample into an embedding mould, adjusting the sample to a preset direction, pouring an embedding agent, so that the fossil sample is wrapped by the embedding agent and bubbles are removed;

Step C2: firstly, yellow light is used for irradiating for 2-4 hours, then ultraviolet light is used for irradiating for 4-6 hours, and after the embedding agent is solidified, the embedding blocks with scales on the surfaces are taken out, so that the fossil sample is directionally embedded.

Further, the embedding mold used in the step C1 comprises a mold groove, wherein the mold groove is formed by downwards sinking a mold plate, and the whole mold groove is in an elongated strip shape; the die groove is provided with a groove bottom surface and four groove side surfaces, and scale protrusions are arranged on the groove bottom surface of the die groove and two of the groove side surfaces which are oppositely arranged, so that the surface of an embedding block formed after the embedding agent is solidified is provided with concave scales.

Further, short scale protrusions are arranged at the positions, close to the right angles, of the bottom surface of the groove, and the short scale protrusions at the two sides of the bottom surface of the groove are discontinuous; the scale bulges on the side surfaces of the grooves are long scale bulges which are vertically and longitudinally arranged.

Further, the number of the short scale protrusions is the same as that of the long scale protrusions, the short scale protrusions and the long scale protrusions are in one-to-one correspondence, and the short scale protrusions and the long scale protrusions are vertically intersected at right angles; or the number of the short scale protrusions is larger than that of the long scale protrusions, and each long scale protrusion perpendicularly intersects with a corresponding short scale protrusion at a right angle.

Further, among the plurality of scale protrusions on the bottom surface of the groove, the lengths of the scale protrusions from the middle to the two sides become shorter in sequence.

Further, the groove side surfaces at the two ends of the length direction of the die groove are arc surfaces, the groove side surfaces at the two ends of the width direction are vertical planes, and the length of the vertical planes is greater than the arc length of the arc surfaces.

Further, the embedding mould is provided with a plurality of mould grooves, and the sizes of the mould grooves are different; the length directions of the plurality of die grooves are arranged in parallel, and the two outermost die grooves are the largest and the same in size.

Further, step D includes the steps of:

step D1: scanning the embedded block again by using 3D-XRM at the resolution of more than 10 mu m for 25+/-5 minutes to obtain low-resolution 3D-XRM data;

step D2: and C, sequentially importing the low-resolution 3D-XRM data and the high-resolution 3D-XRM data obtained in the step B into three-dimensional image processing software, aligning the two groups of data, and acquiring the intersection line of the target to-be-cut surface of the embedding block and the surface of the sample, so as to determine the relative position of the region of interest inside the fossil sample and the scale on the surface of the embedding block.

Further, in step E, scribe positioning is performed on the surface of the embedding block using either of the following two schemes:

the first scheme is as follows: drawing on two adjacent surfaces of the embedding block by using a diamond scribing pen to obtain a junction line;

The second scheme is as follows: laser etching was performed on adjacent two surfaces of the embedded block using a 193nm excimer laser ablation system to obtain a junction line.

Further, in the second scheme, the specific steps include:

step E11: placing a 193nm excimer laser ablation system on the first surface of the embedded sample to be subjected to laser etching upwards;

step E12: opening GeoStar software, and performing optical imaging on the upper surface of the sample by using an optical imaging system;

step E13: determining the positions of points a and b on the embedded sample in the optical photo by using the A, B value obtained in the step D;

step E14: setting ab line segment etching parameters by utilizing the positions of the points a and b on the embedding sample in the steps, and operating software to etch to finish etching of the first marking line;

step E15: taking out the embedded sample, and placing the adjacent second surface upwards into an excimer laser ablation system; and E12-E14 are repeated to obtain the position of the point c, and the laser etching parameters of the bc line segment are set to finish etching scribing positioning and finish etching of the second marking line.

In the step E, when the cutting machine is used for directionally cutting the embedded block, a cutting clamp is used for clamping and fixing the embedded block; the cutting fixture comprises a base body, a fixed connecting piece, an arc-shaped track, an end head and a chuck; wherein the seat body can be connected to the cutting machine; the fixed connecting piece is in sliding connection with the base body, and after the fixed connecting piece and the base body are relatively moved in place, the fixed connecting piece and the base body can be fixed; the convex surface of the arc-shaped track is connected with the fixed connecting piece; the end head is movably connected with the concave surface of the arc-shaped track; the chuck is rotationally connected with the end head, and the chuck rotates on the end head through a rotation adjusting piece arranged on the end head.

Further, in the step F, the focused ion beam carried by the FIB-SEM and Atlas5 software are used for further exposing or polishing the ROI, and the method specifically comprises the following steps:

step F1: adhering the sheet containing the ROI on a 45-degree pre-inclined aluminum nail table;

step F2: placing the aluminum nail table with the adhered sheet into a coating instrument, coating the film, and then placing the coated aluminum nail table into an FIB-SEM;

step F3: tilting the sample holder/aluminum nail table by 45 degrees to enable the sample holder/aluminum nail table to be parallel to the ion beam machining direction, and shooting SEM images; if the ROI is exposed, performing the direct step F6; if the surface is close to the ROI area, sequentially performing the steps F4-F6;

step F4: the sample surface SEM image is initially aligned with the 3D-XRM image;

step F5: ion beam cutting is carried out on the position corresponding to the internal ROI; step F6: the SEM data exposing the ROI are realigned with the 3D-XRM data to ultimately determine the three-dimensional reconstruction region of the sample.

Compared with the prior art, the method for trans-scale comprehensive analysis of the internal structure of the fossil sample has at least one of the following beneficial effects:

1. the method can be used for rapidly, economically and effectively positioning the sample to the position of the region of interest in the fossil sample, and carrying out three-dimensional analysis on the structure and components in the sample; and the image data of the macroscopic three-dimensional space structure is highly correlated with the fine structure and the component information data of the local layer, so that richer and comprehensive scientific information is obtained.

2. The improved embedded grinding tool is adopted, the manufactured embedded block is provided with the scale marks with specific marks, so that the scale marks are convenient to compare with the 3D-XRM, and preparation is made for carrying out surface marking and positioning on the embedded block subsequently; the prepared embedding block is in a long and thin strip shape, so that the clamping and cutting of a cutting machine are facilitated, the distance between a sample and a lens in the 3D-XRM can be reduced, and the scanning resolution is ensured.

3. When in cutting, an improved cutting clamp is adopted, the whole clamp is fixed on the cutting machine after the included angle between the base body and the cutting machine is adjusted, and the position of the fixed connecting piece relative to the base body can be adjusted by moving the fixed connecting piece; the end head slides on the arc-shaped track, so that the rotation angle of the end head relative to the fixed connecting piece can be adjusted, and the angle of the fossil sample can be finely adjusted; the rotary adjusting piece is controlled to rotate the chuck, and the chuck rotates on the end head, so that the angle of the chuck can be adjusted, the angle of the sample is finely adjusted, and the exposure precision of the fossil sample is remarkably improved.

In the invention, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.

Drawings

The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to refer to like parts throughout the several views.

FIG. 1A is a 3D-XRM image of embryonated fossils three-dimensional reconstruction;

FIG. 1B is a slice view derived from a three-dimensional reconstructed 3D-XRM image, with nuclei of interest within the dashed box;

FIG. 2A is a top view of an embedding mold according to an embodiment of the present invention;

FIG. 2B is a schematic view of an embedding mold according to a first embodiment of the present invention;

FIG. 2C is a schematic view of a second angle of an embedding mold according to an embodiment of the present invention;

FIG. 2D is a schematic cross-sectional view of an embedding mold according to an embodiment of the present invention;

FIG. 3 is a schematic illustration of marking indicia on the surface of an embedded sample in accordance with an embodiment of the present invention;

FIG. 4 is an SEM image of nuclei in embryonic cells after ion beam processing;

FIG. 5A is an SEM image of the nuclear sites after carbon and platinum deposition and labeling;

FIG. 5B is an SEM image of a region of interest after U-grooving;

FIG. 6 is an image of a spatial distribution reconstruction model of elements obtained based on FIB-SEM superimposed on a three-dimensional reconstruction model obtained based on FIB-SEM in VGstudio Max software;

FIG. 7 is an image of a spatially distributed model of elements (small field of view) based on FIB-SEM superimposed on a three-dimensional structural model (large field of view) based on 3D-XRM in VGstudio Max software;

FIG. 8 is a schematic view of a first angle configuration of a cutting fixture according to an embodiment of the present invention;

FIG. 9 is a schematic view of a second angular configuration of a cutting fixture in accordance with an embodiment of the present invention;

FIG. 10 is a schematic illustration of a cutting fixture with parts separated in an embodiment of the present invention.

Reference numerals:

100. embedding glue; 200. a region of interest in the sample; 300. fossil samples; 400. a first mark line; 500. a second mark line;

1. a mold plate; 2. a bottom surface of the groove; 3. a side surface of the long groove; 4. short groove side surfaces; 5. a long scale protrusion; 6. short scale protrusions;

10. a base; 101. a cross beam; 102. a longitudinal beam; 103. ball head; 104. a sleeve;

20. fixing the connecting piece; 201. a side foot; 2011. a fixing through hole; 2022. a set screw; 202. a connecting block; 203. a sliding table; 2031. a slide hole; 2032. abutting the screw; 204. a top connection hole; 205. a side screw; 206. inserting a connecting rod;

30. an arc-shaped track; 301. a first angle adjusting lever;

40. an end head; 401. a second angle adjusting lever;

50. A chuck.

Detailed Description

The following detailed description of the preferred invention is provided in connection with the accompanying drawings, which form a part hereof, and together with the description serve to explain the principles of the invention, and are not intended to limit the scope of the invention.

Example 1

The invention discloses a method for trans-scale comprehensive analysis of an internal structure of a fossil sample, in particular to a method for combining trans-scale three-dimensional space structure image and component information of the internal structure of the fossil sample, which comprises the following steps:

step A: fossil samples to be analyzed are screened using an optical microscope (LM) or a Scanning Electron Microscope (SEM).

And (B) step (B): scanning the selected fossil sample in a high resolution scanning mode by using the 3D-XRM to obtain high resolution 3D-XRM data; and performing three-dimensional reconstruction based on the 3D-XRM data to obtain an internal structural image of the fossil sample, and determining an internal region of interest (ROI) of the fossil sample.

Step C: and manufacturing the selected fossil sample into an embedding block with scale marks on the surface.

Step D: using 3D-XRM to rapidly scan the embedded block in a low resolution mode to obtain low resolution 3D-XRM data; and aligning the low-resolution data obtained in the step with the high-resolution 3D-XRM data obtained in the step B in three-dimensional image processing software, and determining the relative position of the region of interest inside the fossil sample and the scale on the surface of the embedding block.

Step E: marking and positioning on the surface of the embedded block based on the determined relative positions of the region of interest inside the fossil sample and the scales on the surface of the embedded block so as to determine a surface to be cut; and directionally cutting the embedding block along the surface to be cut by using a cutting machine to obtain a sheet containing the ROI, and grinding one of the cutting surfaces to obtain the sheet with the ROI exposed.

Step F: the ROI is further exposed or polished.

Step G: and performing test analysis on the thin sheet exposed with the ROI by using the FIB-SEM, such as secondary electron/back scattering scanning, EBSD/EDS and other two-dimensional/three-dimensional analysis, and introducing the obtained FIB-SEM three-dimensional data into VGstudio Max software for three-dimensional reconstruction.

Step H: and (3) introducing the high-resolution 3D-XRM data into VGstudio Max software containing the FIB-SEM three-dimensional data, and performing data processing and fine alignment to obtain a cross-scale multi-mode analysis result of the same fossil by combining the two technologies. Wherein, the trans-scale refers to different resolutions or field sizes, and the multi-mode refers to different data types.

Specifically, the step a includes the following steps:

step A1: fossil sample screening, wherein the phosphated embryo-like fossil is subjected to acid soaking, and then is subjected to manual screening under a stereo microscope, so that the fossil is separated from insoluble rock residues. If the preservation quality of the fossil sample can be assessed under a stereoscopic microscope, step B can be performed. Otherwise, step A2 is performed.

Step A2: SEM was used for additional screening. Finally, fossil samples with better preservation quality are selected for subsequent experimental analysis.

Specifically, the step B includes the following steps:

step B1: and scanning the target fossil sample in a high-resolution scanning mode by using the 3D-XRM to obtain high-resolution 3D-XRM data, and accurately positioning the ROI based on the high-resolution 3D-XRM data. The high resolution refers to resolution of 0.5-3 mu m, the high resolution scanning time is 3-8 hours, and the high resolution three-dimensional structure data of the fossil sample can be obtained through data processing. Since fossil samples are small, during the experiment, the operating voltage of the X-ray tube is set to 50 kv for optimum image contrast, and a 4X/10X objective is used to select the appropriate exposure time for a single projection, e.g. set to 6 seconds. To mitigate artifacts, LE2 filters are used to filter low energy X-ray wavelengths. In the scanning process, the sample rotates 360 degrees, and the number of projection images acquired by the imaging system is set according to the requirement.

Step B2: and B1, performing data processing (cutting, filtering, denoising and the like) and three-dimensional reconstruction on the high-resolution 3D-XRM data obtained in the step B1 on VGstudio Max software to obtain the volume data of the fossil sample. The volume data is exported in the form of a stack of virtual slice images.

Step B3: based on the obtained fossil sample volume data, the volume data is subjected to segmentation, rendering, color configuration and other necessary processing to obtain three-dimensional visual expression results of the fossil sample, such as mesh files, animations and the like.

In this example, FIG. 1A shows a three-dimensional reconstructed 3D-XRM image of an embryonated fossil sample, revealing that the sample consists of several polyhedral shaped cells. In addition, each cell contains a spherical nucleus, as shown in FIG. 1B, which is a slice derived from a three-dimensional reconstructed 3D-XRM image, each nucleus consisting of two different mineral phases. The grey values of the two mineral phases are obviously different; however, the specific mineral type cannot be distinguished only from the tomographic image data. To obtain the exact composition of these two mineral phases, we selected one nucleus located within one cell as our subject of investigation, the nucleus of interest was located within the dashed box of fig. 1B, and prepared for three-dimensional EDS analysis to obtain spatial distribution information of the elements as well as finer structural data.

The nuclei selected here act as ROIs, and since the nuclei selected are located within the phosphorylated cytoplasm, rather than exposed to the cell surface, direct elemental detection using EDS is not feasible. Thus, the sample needs to be processed to expose the ROI.

Precise exposure of the ROI is a challenging task, in which high resolution 3D-XRM data of the sample as a whole is critical to navigating to accurately locate the ROI. If the observed ROI is less than or equal to 100 μm from the upper surface of the sample, the FIB (focused ion beam) is used for processing, and the exposure and positioning of the ROI are performed. If the observed ROI is at a distance > 100 μm from the upper surface of the sample, steps C-E are used for ROI exposure and repositioning.

Specifically, in the step C, the fossil sample is directionally embedded by using an embedding mold with scale protrusions to obtain an embedding block containing the fossil sample, so that the surface of the embedding block is provided with scale marks with pits, and the method specifically comprises the following steps:

step C1: and (3) selecting an embedding mould with a proper size, placing the fossil sample into the embedding mould, adjusting the sample to a preset direction, pouring an embedding agent, enabling the fossil sample to be wrapped by the embedding agent, and vacuumizing to remove bubbles in the fossil sample. Among them, the embedding agent is preferably a Technovit 7200VLC single-component resin embedding agent. When the sample is adjusted to a predetermined direction, the ideal cross section of the ROI can be determined based on the step B, so that the ideal cross section of the ROI is perpendicular to the long side groove side surface of the mold groove as much as possible when the sample is embedded. The long strip embedding block with scales on the surface is formed after the long strip embedding block is solidified in the mode, wherein the ideal section of the ROI is vertical or approximately vertical to the side face of the long groove of the embedding block, so that the cutting efficiency is improved, and the loss of a sample is reduced. The angle between the ideal cross section of the ROI and the long wall surface of the embedded block is in the angle adjustment range of the EXAKT EP300 cutting machine.

Step C2: firstly, yellow light is used for irradiating for 2-4 hours, then ultraviolet light is used for irradiating for 4-6 hours, and after the embedding agent is solidified, the embedding blocks with scales on the surfaces are taken out, so that the fossil sample is directionally embedded.

In this embodiment, the difference between the step D and the step B is that the scan field and the scan speed are different. The scanning in the step B is aimed at fossil samples, and high-resolution internal structure images of the samples can be obtained. Step D is to obtain the relative positions of the embedded block scales and the outline of the fossil sample, and the resolution is not high. Such a solution design improves the scanning efficiency. Specifically, the step D includes the steps of:

step D1: the embedded block was again rapidly scanned in low resolution mode using 3D-XRM to obtain low resolution 3D-XRM data. Wherein, the low resolution is the resolution of more than 10 μm, and the scanning time is 25+ -5 minutes.

Step D2: sequentially importing the obtained low-resolution 3D-XRM data and the high-resolution 3D-XRM data obtained in the step B into three-dimensional image processing software, aligning the two groups of data by utilizing information such as a sample contour and the like, and acquiring intersection lines of a target surface to be cut of an embedding block and the surface of the sample in the three-dimensional image processing software, such as ab and bc line segments in FIG. 3, so as to determine the relative positions of an interested region inside a fossil sample and scales on the surface of the embedding block; fig. 3 shows the positional relationship of the cured embedding glue 100, the region of interest 200 in the sample, the fossils sample 300, and the first marker line 400 and the second marker line 500, wherein the first marker line 400 corresponds to the line segment ab and the second marker line 500 corresponds to the line segment bc. Three pitch values are obtained in three-dimensional image processing software using a metrology tool: A. b, C. Wherein the graduation marks serve as characteristic starting points for easy identification.

In the step E, the following two schemes are utilized to carry out marking and positioning on the surface of the embedded block, two marking lines are respectively positioned on two adjacent surfaces of the embedded block, and the plane where the two marking lines are positioned is the surface to be cut:

first scribing and positioning scheme: the first marking line 400 and the second marking line 500 are respectively marked on two adjacent surfaces of the embedding block according to the position of the intersection line by using a diamond marking pen.

The second scribing and positioning scheme is as follows: and (3) performing laser etching scribing positioning on two adjacent surfaces of the embedded block by using a 193nm excimer laser ablation system, and performing laser etching to obtain the first marking line 400 and the second marking line 500.

When the second scribing positioning scheme is implemented, the method specifically comprises the following steps:

step E11: the first surface of the embedded sample to be subjected to laser etching is upward, and the embedded sample is placed in a 193nm excimer laser ablation system, wherein the 193nm excimer laser ablation system adopts a RESOUTION SE-S155 type.

Step E12: the GeoStar software was opened and the upper surface of the sample was optically imaged using an optical imaging system using a 100X lens.

Step E13: based on the position of the intersection line, using the A, B value obtained in step D, the GeoStar software measurement was used to determine the position of points a, b on the embedded sample in the optical photograph.

Step E14: and setting ab line segment etching parameters by utilizing the positions of the points a and b on the embedding sample in the steps, and running software to perform the etching to finish the etching of the first marking line 400. The laser etching parameters are as follows: the diameter of the beam spot is 5-10 mu m, the repetition rate is 100-300Hz, the scanning speed is 0.2mm/s, and the fluence is 16J.cm ² 。

Step E15: taking out the embedded sample, and placing the adjacent second surface needing laser etching upwards into an excimer laser ablation system. And E12-E14 are repeated to obtain the position of the point c, laser etching parameters of the bc line segment are set to finish etching scribing positioning, etching of the second marking line 500 is finished, and the surface to be cut is determined based on the intersected first marking line 400 and second marking line 500.

In step E, after finishing scribing and positioning the surface of the embedding block, determining a surface to be cut, performing directional cutting on the embedding block along the surface to be cut by using a cutting machine to obtain a slice containing the ROI, and grinding one of the cutting surfaces to obtain a slice exposing the ROI, which specifically comprises the following steps:

step E21: an EXAKT EP300 cutter is used to cut directionally along the surface to be cut to expose or approximate the ROI. Specifically, a cutting clamp is used for clamping and fixing an embedded block positioned by etched scribing, the angle and the height of the embedded block are adjusted to a proper space position and posture, the laser of an EXAKT EP300 cutting machine is turned on, so that the laser of the cutting machine can approach or basically align to a surface to be cut, after the laser is basically aligned, the laser is further adjusted to a required cutting surface by adjusting the stepping of the cutting machine, the minimum precision of the stepping adjustment is 1 mu m, the EXAKT EP300 cutting machine is started, and the diamond band saw performs directional cutting along the surface to be cut to obtain a first cutting surface. And continuously adjusting the stepping of the cutting machine, cutting off the bottom of the sample to obtain a second cutting surface parallel to the first cutting surface, and finishing the cutting preparation of the thin sheet containing the ROI, wherein the first cutting surface and the second cutting surface are two surfaces of the thin sheet, and the distance between the two surfaces is the thickness of the thin sheet.

Step E22: after directional cutting is completed to obtain a sheet containing the ROI, grinding the first cutting surface by using an EXAKT 400CS hard tissue grinding machine to obtain a sheet exposing the ROI, and the method comprises the following steps of:

a second cut surface of the ROI-containing sheet was adhered to a slide. The distance of the first cutting plane from the mark lines ab, bc is observed under a microscope. The slide was mounted to an EXAKT 400CS hard tissue lapping machine. And selecting a proper number of sand paper, such as 300-4000 meshes, and setting the automatic grinding thickness of the grinding disc machine for further grinding, wherein the gradient selection can be realized. And observing the cut surface of the sample under a microscope, and if the ROI is not exposed, repeating the grinding step, and continuing grinding to approach or reach the position of the ROI to obtain a grinding and polishing surface. It should be noted that this process requires frequent observation of the polished cut surface in order to prevent the region of the ROI from being missed due to too fast polishing. If a complete three-dimensional reconstruction of the region of interest sample is desired, it is sometimes desirable to preserve a partial region of the upper surface of the ROI in order to prevent damage to the ROI. If the ROI is located too deep in the sample, the exposure by direct FIB sputtering is time consuming and inefficient, and the directional embedding, cutting and grinding procedures of this embodiment are more efficient.

Specifically, step F uses a FIB-SEM-mounted focused ion beam and Atlas5 software to further expose or polish the ROI, comprising the steps of:

step F1: the sheet containing the ROI was glued to a 45 degree pre-tilted aluminum stapling table. One of the two surfaces of the sheet, which is closer to the ROI, is a polished surface, and the other surface is a bonding surface bonded to an aluminum nail table.

Step F2: and (3) placing the aluminum nail table with the thin sheet into a film plating instrument, coating a gold film of 20-100nm on the surface of the thin sheet to enhance the conductivity of the sample, taking out, placing the sample on a FIB-SEM sample holder, and placing the sample in the FIB-SEM.

Step F3: the sample holder/aluminum stage was tilted 45 degrees parallel to the ion beam machining direction and SEM images were taken using Atlas5 software. If the ROI has been exposed, a direct step F6 is performed. If the surface has approached the ROI area, steps F4-F6 are performed sequentially.

Step F4: the sample surface SEM images were initially aligned with the 3D-XRM images. Specifically, the internal position of the ROI in the sample, including the relative position of the ROI and xy plane, and the depth from the surface, is confirmed by high resolution 3D-XRM images after alignment.

Step F5: ion beam cutting is performed on the position corresponding to the internal ROI. The relative position of the ROI in the xy plane in the above step, and the depth from the surface are used as references to determine the position and depth to be sputtered. The voltage beam current of the ion beam was 30kV,65nA. With the progressive exposure of the ROI, it is necessary to reduce the ion beam current for finer processing. The gradient was surface finished using a small ion beam such as 30nA, 15nA, 7nA, 1.5nA, etc. The cut surface of the ion beam cut nuclear position is shown in fig. 4.

Step F6: the SEM data exposing the ROI are realigned with the 3D-XRM data to ultimately determine the three-dimensional reconstruction region of the sample. Specifically, atlas5 software was used for manual alignment of virtual slice data images of ROIs on sample global structure data and SEM images obtained by 3D-XRM. This step is also advantageous for fine data alignment and combination of step H.

After successful exposure of the ROI, the next step is three-dimensional reconstruction of the structural and elemental composition of the ROI using FIB-SEM.

Specifically, step G comprises the steps of:

step G1: FIB-SEM accurately deposits carbon and platinum layers on the selected ROI surface by means of ion beam induced deposition functions, i.e. deposition layers using trimethylboron and methylcyclopentadienyl platinum as organometallic precursors, as shown in fig. 5A. Has the following functions: 1) The deposited layer protects the ROI from damage during subsequent ion beam sputtering. 2) The designed contrast-differentiated reticle is used for auto-focusing of SEM and determining slice thickness in auto-three-dimensional reconstruction. 3) Two asterisk marks were designed for spectral imaging autofocus.

Step G2: after carbon/platinum deposition is completed, a U-shaped trench is cut around the ROI as shown in fig. 5B. The FIB-SEM then automatically performs ion beam sputtering and SEM imaging according to preset parameters. To collect the elemental distribution information of the ROI, the FIB-SEM system invokes EDS to image the elemental composition of the ROI while collecting the SEM images. In order to obtain high quality images and reduce the charging effect, the electron beam working voltage is 1-5kV, and the beam current is 500pA-1nA. In the SEM-EDS analysis process, the working voltage of the electron beam is automatically adjusted to 8-15kV so as to ensure the generation of strong X-ray signals.

In this example, the Ga+ focused ion beam has an operating voltage of 30kV and a beam current of 1.5nA. For each cross section (slice), the SEM collects two sets of image data for Secondary Electron (SE) and backscattered electron (BSE) modes. In this experiment the z-axis resolution was set to 10nm and a total of 2614 slice images were acquired with a reconstructed volume of ROI of approximately 102.4 x 30 x 26.2 μm. EDS (oxford ulmmmax 170) performs elemental imaging once every 10 slices, producing 237 elemental imaging maps in total.

Step G3: the result obtained in the above steps is a three-set image stack comprising Secondary Electron (SE), backscattered electron (BSE) images and elemental distribution maps of a series of slices. The image stack may be three-dimensionally reconstructed and analyzed using volume data processing software (e.g., vgstudomax). Since the back-scattered electron images provide superior contrast in reflecting mineral phase differences, the fine three-dimensional structure of the ROI is reconstructed by superimposing the secondary electron images with the back-scattered electrons. This reconstruction may optionally be performed in vgstadium. At the same time, the element profile of the ROI is imported into vgstadiomax, and the reconstructed element spatial distribution model is superimposed on the three-dimensional structure model (fig. 6).

In the case of local-level (nano-level) component analysis of micrometer-scale and larger-scale objects, it is often difficult to determine the original orientation of the cutting layer in the sample, which is highly likely to cause interpretation bias or misjudgment. In this embodiment, since the ROI represents only a small portion of the nucleus, it is challenging to understand the morphology of the entire nucleus by examining only the structure and composition of the ROI. To solve this problem, the three-dimensional structural model and the three-dimensional element distribution model of the ROI must be integrated into the nuclear 3D-XRM model of a wider field of view and finely aligned. Therefore, the data isolation between the devices is broken, and the macroscopic three-dimensional space structure image data and the local layer composition information data are overlapped and reproduced.

Specifically, the step H includes the steps of:

step H1: the high resolution 3D-XRM data is imported into vgstadiomax software in the above steps.

Step H2: manual calibration is performed based on easily identifiable structural features such as cell wall ravines. This step involves fine alignment of the x, y, z data, and eventually the local cell wall of the ROI with a body resolution of 10nm can be combined with the nuclear model into an embryonic fossils model with a body resolution of 1.3 μm, as shown in fig. 7. The local cell wall and the cell nucleus model of the ROI are small-view high-resolution energy spectrum data, and the integral embryo fossils model is large-view low-resolution 3D-XRM structure data. The analysis result of the same fossil cross-scale (different resolution/visual field size) -multi-mode (different data types) by combining the two technologies is obtained.

Compared with the prior art, the cross-scale comprehensive analysis method for the internal structure of the fossil sample has the following beneficial effects:

a) The method realizes the combination of the 3D-XRM and the FIB-SEM, the large-field volume data of the 3D-XRM provides effective navigation, the exposure efficiency of the region of interest in the fossil sample is effectively improved through the directional embedding and the accurate cutting of the sample, the data combination precision is improved, the positioning to the region of interest in the sample is facilitated to carry out the rapid comprehensive analysis, the sample processing and preparation cost is reduced, the loss of rare samples is reduced, and the experimental efficiency is improved.

b) The combined 3D-XRM and FIB-SEM test has the following advantages: firstly, accurately positioning an interested region under large-field data with micron resolution, and realizing efficient experimental operation to obtain key data of the interested region; secondly, for a large-area structure with homogeneity, the whole structure can be reversely deduced by using three-dimensional micro-area data with nanometer resolution, so that the analysis resolution is improved; third, misjudgment and multiple solutions caused by information insufficiency are eliminated.

c) Embedding is carried out on a fossil sample by adopting an embedding mould with scales, and sample cutting and grinding treatment is carried out by matching with a cutting and grinding system, so that the positioning information identification sharing of the 3D-XRM and the FIB-SEM equipment on the region of interest in the same object can be realized, thus obtaining three-dimensional data information of the sample in a trans-scale (different resolutions) -multi-mode (different data types), including 3D-XRM structure information of micron-level resolution, SEM morphology information of nanometer-level, abundant EDS component information/EBSD crystal structure information and the like, and improving the capability of scientific researchers to collect the internal structure and component information of the fossil at the same time. Moreover, the embedded die is simple in structure, the fossil sample can be processed by adopting a common cutting machine, the operation is convenient, the cost is low, and the efficiency is greatly improved compared with the traditional random polishing.

Example 2

In yet another embodiment of the present invention, an embedding mold is disclosed for preparing an embedding block with scale marks on the surface in step C of embodiment 1.

As shown in fig. 2A to 2D, the embedding mold includes a mold groove formed by downwardly recessing the mold plate 1, and the mold groove is formed in an elongated strip shape as a whole; the die groove is provided with a groove bottom surface 2 and four groove side surfaces, and scale protrusions are arranged on the groove bottom surface 2 of the die groove and two of the groove side surfaces which are oppositely arranged, so that the surface of an embedding block formed after the embedding agent is solidified is provided with concave scales.

Compared with the prior art, the embedded grinding tool provided by the embodiment has the advantages that the structure is simple, the operation is convenient, the manufactured embedded block is provided with the scale marks with specific marks, the comparison with the 3D-XRM is convenient, and the preparation is made for the subsequent surface marking and positioning of the embedded block; the prepared embedding block is in a long and thin strip shape, so that the clamping and cutting of a cutting machine are facilitated, the distance between a sample and a lens in the 3D-XRM can be reduced, and the scanning resolution is ensured.

In some embodiments, the width of the graduation projections is 100 μm, the height is 20 μm, the distance between two adjacent graduation projections on the bottom surface 2 of the groove is 1mm, and correspondingly, the width of the graduation on the surface of the prepared embedding block is 100 μm, the depth is 50 μm, and the distance between two adjacent graduation marks is 1mm.

In some embodiments, the position of the bottom surface 2 of the groove close to the right angle is provided with short scale protrusions 6, the short scale protrusions 6 on two sides of the bottom surface 2 of the groove are not continuous, the short scale protrusions 6 on two sides of the bottom surface 2 of the groove are disconnected, the scale protrusions on the side surface of the groove are long scale protrusions 5 which are vertically arranged in a through length mode, the length of the long scale protrusions 5 on the side surface of the groove is the same as the groove depth of the groove of the die, namely, the length of the long scale protrusions 5 on the side surface of the groove is the same as the height of the side surface of the groove. If the long scale protrusions 5 on the sides of the groove are interrupted, the sample embedding block cannot be pulled out, so the scale protrusions on the sides of the groove are designed to extend from the bottom to the top. The structural design not only ensures that the scale on the embedded block can be identified on a finer basis, but also can easily separate the embedded block from the embedding mould.

In an alternative embodiment, the number of short scale protrusions 6 is the same as the number of long scale protrusions 5, i.e. the short scale protrusions 6 are in one-to-one correspondence with the long scale protrusions 5 and meet vertically at right angles.

In another alternative embodiment, the number of short scale protrusions 6 is greater than the number of long scale protrusions 5, each long scale protrusion 5 meets a corresponding short scale protrusion 6 vertically at a right angle, for example, one short scale protrusion 6 is arranged between two adjacent long scale protrusions 5, the distance between two adjacent long scale protrusions on the side surface of the groove is twice the distance between two adjacent short scale protrusions on the bottom surface of the groove, and one long scale line is arranged every 2mm on the side wall of the prepared embedding block for easy identification.

In some embodiments, the lengths of the scale protrusions on the bottom surface 2 of the groove from the middle to the two sides are sequentially shortened, that is, the scale protrusion on the bottom surface 2 of the groove is longest, and the longest scale mark on the embedding block is taken as a zero point, so that the zero point of the scale can be conveniently identified.

In some embodiments, the groove sides at the two ends of the length direction of the mold groove are arc surfaces, the groove sides at the two ends of the width direction are vertical planes, and the length of the vertical planes is greater than the arc length of the arc surfaces. It is also understood that there are two long groove sides 3 and two short groove sides 4 in the four groove sides of the mold groove, the long groove sides 3 are two parallel vertical planes, and the short groove sides 4 are two vertically arranged arc surfaces. The embedding blocks are convenient to separate out by arranging the two ends in the length direction as the side faces of the arc-shaped grooves.

In some embodiments, the embedding mold has a plurality of mold grooves, the mold grooves being sized differently to accommodate samples of different sizes. The length directions of the plurality of mold grooves are arranged in parallel, the sizes of the two outermost mold grooves are the largest and the same, and the sizes of the plurality of mold grooves in the middle are smaller than the sizes of the outermost mold grooves, so that the embedding mold can be operated on the test bed in a balanced manner.

In some embodiments, the mold plate 1 is an elliptical plate, the mold groove is located in the middle area of the elliptical mold plate 1, the elliptical outer frame formed by the edge of the mold plate 1 is used for supporting, and can be matched with the light-curing embedding instrument, so that when the embedding mold is placed into the light-curing groove of the light-curing embedding instrument, the bottom is suspended for cooling, and the outer frame plays a supporting role.

Example 3

In still another embodiment of the present invention, a directional cutting jig for embedding fossil samples, hereinafter referred to as "cutting jig" for short, is disclosed for clamping and fixing an embedding block while performing directional cutting along a surface to be cut with a cutting machine in step E of embodiment 1.

Referring to fig. 8 to 10, the cutting jig includes a base 10, a fixing connector 20, an arc-shaped rail 30, a head 40, and a collet 50; wherein, the base body 10 can be connected with a cutting machine, and the cutting machine can adopt an EXAKT EP300 type cutting machine; the fixed connecting piece 20 is in sliding connection with the seat body 10, and after the fixed connecting piece 20 and the seat body 10 relatively move in place, the fixed connecting piece 20 and the seat body can be fixed; the convex surface of the arc-shaped track 30 is connected with the fixed connecting piece 20; the end 40 is movably connected with the concave surface of the arc-shaped track 30; the collet 50 is rotatably coupled to the head 40 and the collet 50 rotates on the head 40 via a rotation adjustment member provided on the head 40.

When the fixture is implemented, after the chuck 50 clamps a sample, the whole fixture is fixed on the cutting machine by adjusting the included angle between the ball head 103 and the cutting machine, the included angle between the base body 10 and the z-axis is adjusted to be plus or minus 20 degrees, and the position of the fixed connecting piece 20 relative to the base body 10 can be adjusted by moving the fixed connecting piece 20; the end 40 slides on the arc-shaped track 30, the rotation angle of the end 40 relative to the fixed connecting piece 20 can be adjusted, the included angle between the end 40 and the z axis of the clamp can be adjusted by plus or minus 25 degrees longitudinally, so that the angle of a fossil sample can be finely adjusted, the rotating adjusting piece is controlled to rotate the clamping head 50, the clamping head 50 rotates on the end 40, the angle of the clamping head 50 can also be adjusted, the clamping head 50 can rotate in the xy plane by 360 degrees, the angle of the sample can be finely adjusted, and the included angle range between the clamp and the z axis is plus or minus 45 degrees, namely the adjustable range of 90 degrees in total. The base 10, the fixed connector 20, the end 40 and the chuck 50 are sequentially arranged along the z-axis direction, and the xy plane is perpendicular to the z-axis.

Compared with the prior art, the directional cutting clamp for embedding the fossil sample has the advantages that after the included angle between the base body and the cutting machine is adjusted, the whole clamp is fixed on the cutting machine, and the position of the fixed connecting piece relative to the base body can be adjusted by moving the fixed connecting piece; the end head slides on the arc-shaped track, so that the rotation angle of the end head relative to the fixed connecting piece can be adjusted, and the angle of the fossil sample can be finely adjusted; the rotary adjusting piece is controlled to rotate the chuck, and the chuck rotates on the end head, so that the angle of the chuck can be adjusted, the angle of the sample is finely adjusted, and the exposure precision of the fossil sample is remarkably improved.

In some embodiments, the seat body 10 comprises a cross beam 101, a longitudinal beam 102, a ball head 103 and a sleeve 104, the cross beam 101 is connected to the longitudinal beam 102 in a penetrating way, the cross beam 101 comprises two cross beams 101 which are arranged in parallel, and the overall construction stability and strength are better; the bulb 103 is connected at the lateral wall of longeron 102, and the tip of sleeve 104 is equipped with the trepanning, and bulb 103 passes the trepanning and extends to in the sleeve 104, and bulb 103's head diameter is greater than the diameter of trepanning, and bulb 103 can move relatively, and the lateral wall of sleeve 104 is equipped with the screw thread. The sleeve 104 is connected with the corresponding part of the cutting machine, and the ball head 103 can rotate by an angle of plus or minus 20 degrees relative to the sleeve 104 with the z-axis being 0 degrees. The head diameter of the ball 103 is larger than the diameter of the trepanning so that the ball 103 and the sleeve 104 will not disengage.

In some embodiments, the fixed connector 20 includes a side leg 201 and a connecting block 202, the side leg 201 is connected to two sides of the connecting block 202, the side leg 201 is connected with a sliding table 203, and the sliding table 203 is slidably connected with the cross beam 101.

The sliding table 203 is arranged to enable the whole connecting block 202 to slide on the cross beam 101, so that the clamping head 50, the end head 40 and the fixed connecting piece 20 can move relative to the base body 10.

The fixed connector 20 formed by the side legs 201 and the connecting blocks 202 is in a trapezoid block structure. A fixed connector 20 is provided to connect the base 10 with the head 40 and the collet 50.

The slipway 203 wears to locate the crossbeam 101, and slipway 203 includes two, is connected to the side leg 201 respectively, and two slipways 203 are located the both sides of longeron 102 respectively.

One end of the sliding table 203 has a sliding hole 2031, an abutment screw 2032 is screwed into the sliding hole 2031, and the abutment screw 2032 can abut against the cross beam 101 in the sliding table 203.

After the slide table 203 is slid to a predetermined position, the abutment screw 2032 can be rotated, and the abutment bolt abuts against the cross beam 101 in the slide table 203, thereby fixing the slide table 203 to the cross beam 101.

The side leg 201 is provided with a fixing through hole 2011, the fixing through hole 2011 is provided with a fixing screw 2022, the sliding table 203 is provided with a corresponding fixing groove, and the fixing screw 2022 can be connected into the fixing groove in a threaded manner to fix the side leg 201 and the sliding table 203. The fixing screw 2022 is provided, the fixing screw 2022 can be screwed into the fixing through hole 2011 and the fixing groove, the side leg 201 and the sliding table 203 are connected, and the fixing screw 2022 can be detached.

In some embodiments, the top of the fixed connector 20 is provided with a top connecting hole 204, the side surface of the fixed connector 20 is provided with a side surface screw 205, the side surface screw 205 is penetrated into the top connecting hole 204 through a side surface screw hole formed in the side surface of the fixed connector 20, the bottom of the arc track 30 is connected with a plugging rod 206, the plugging rod 206 is inserted into the top connecting hole 204, the side wall of the plugging rod 206 is provided with a plugging screw hole corresponding to the side surface screw hole, and the side surface screw 205 penetrates through the side surface screw hole to be connected into the plugging screw hole in a threaded manner. After the plugging rod 206 at the bottom of the arc-shaped track 30 is inserted into the top connecting hole 204 of the fixed connecting piece 20, the side screw 205 is rotated into place, and the side screw 205 is screwed into the plugging threaded hole, so that the plugging rod 206 and the fixed connecting piece 20 can be fixedly connected.

The top connecting hole 204 and the side threaded hole are both arranged on the connecting block 202, the top connecting hole 204 is arranged at the top of the connecting block 202, and the side threaded hole is arranged on the side of the connecting block 202.

In some embodiments, the bottom of the end 40 has a groove, the arc track 30 is clamped with the groove, a rotation hole is formed in the side surface of the end 40, the rotation hole is communicated with the groove, a first angle adjusting rod 301 is rotationally connected in the rotation hole, one end of the first angle adjusting rod 301 located in the groove is connected with a first gear, an arc tooth slot is formed in the side surface of the arc track 30, and the first gear is meshed with the arc tooth slot.

Rotating the first angle adjustment lever 301 causes the first gear to rotate and the head 40 as a whole moves on the arcuate track 30, thereby producing an angular change. The end 40 slides on the arc-shaped track 30, so that the rotation angle of the end 40 relative to the z-axis can be adjusted, and the included angle between the end 40 of the clamp and the z-axis can be longitudinally adjusted by plus or minus 25 degrees, thereby finely adjusting the angle of the fossil sample

In some embodiments, the side of the end 40 opposite to the rotating hole is provided with an angle hole, a second angle adjusting rod 401 is arranged in the angle hole, the top of the end 40 is provided with an angle groove, the chuck 50 is rotationally connected in the angle groove, the angle hole is communicated with the angle groove, the second angle adjusting rod 401 is arranged in the angle hole in a penetrating way and extends into the angle groove, the end part of the second angle adjusting rod 401 is connected with a second gear, and the second gear is meshed with a toothed ring at the bottom of the chuck 50.

The second angle adjusting lever 401 is rotated to drive the second gear to rotate, so that the angle of the chuck 50 can be adjusted, and the chuck 50 can be rotated in the xy plane by 360 degrees, thereby finely adjusting the angle of the fossil sample.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims (10)

1. The trans-scale comprehensive analysis method for the internal structure of the fossil sample is characterized by comprising the following steps of:

and (B) step (B): scanning the fossil sample in a high-resolution scanning mode by using the 3D-XRM to obtain high-resolution 3D-XRM data; three-dimensional reconstruction is carried out based on 3D-XRM data to obtain an internal structural image of the fossil sample, and an ROI (region of interest) in the fossil sample is determined;

step C: manufacturing the fossil sample into an embedding block with scale marks on the surface;

step D: using 3D-XRM to rapidly scan the embedded block in a low resolution mode to obtain low resolution 3D-XRM data; and aligning the low-resolution data obtained in the step with the high-resolution 3D-XRM data obtained in the step B in three-dimensional image processing software, and determining the relative position of the region of interest inside the fossil sample and the scale on the surface of the embedding block;

Step E: marking and positioning on the surface of the embedded block based on the determined relative positions of the region of interest inside the fossil sample and the scales on the surface of the embedded block so as to determine a surface to be cut; directionally cutting the embedding block along the surface to be cut by using a cutting machine to obtain a sheet containing the ROI, and grinding one of the cutting surfaces to obtain a sheet exposing the ROI;

step G: performing test analysis on the thin sheet exposed with the ROI by using the FIB-SEM, and importing the obtained FIB-SEM three-dimensional data into VGstudio Max software for three-dimensional reconstruction;

step H: and (3) introducing the high-resolution 3D-XRM data into VGstudio Max software containing the FIB-SEM three-dimensional data, and performing data processing and fine alignment to obtain a cross-scale multi-mode analysis result of the same fossil by combining the two technologies.

2. The method of claim 1, further comprising step F between step E and step G: the ROI is further exposed or polished.

3. The method of claim 1, wherein step B comprises the steps of:

step B1: scanning a target fossil sample with a resolution of 0.5-3 μm by using a 3D-XRM, wherein the scanning time is 3-8 hours, obtaining high-resolution 3D-XRM data, and accurately positioning the ROI based on the high-resolution 3D-XRM data;

Step B2: performing data processing and three-dimensional reconstruction on the high-resolution 3D-XRM data to obtain the volume data of the fossil sample;

step B3: based on the volume data, a three-dimensional visual expression result of the fossil sample is obtained.

4. The method of claim 1, wherein step C comprises the steps of:

step C1: placing the fossil sample into an embedding mould, adjusting the sample to a preset direction, pouring an embedding agent, so that the fossil sample is wrapped by the embedding agent and bubbles are removed;

step C2: firstly, yellow light is used for irradiating for 2-4 hours, then ultraviolet light is used for irradiating for 4-6 hours, and after the embedding agent is solidified, the embedding blocks with scales on the surfaces are taken out, so that the fossil sample is directionally embedded.

5. The method for cross-scale integrated analysis of internal structures of fossil samples according to claim 5, wherein the embedding mold used in step C1 comprises a mold groove formed by downwardly recessing a mold plate, and the mold groove is formed in an elongated strip shape as a whole; the die groove is provided with a groove bottom surface and four groove side surfaces, and scale protrusions are arranged on the groove bottom surface of the die groove and two of the groove side surfaces which are oppositely arranged, so that the surface of an embedding block formed after the embedding agent is solidified is provided with concave scales.

6. The method for cross-scale comprehensive analysis of internal structures of fossil samples according to claim 5, wherein short scale protrusions are arranged at the positions of the bottom surface of the tank close to right angles, and the short scale protrusions at the two sides of the bottom surface of the tank are discontinuous; the scale bulges on the side surfaces of the grooves are long scale bulges which are vertically and longitudinally arranged.

7. The method for cross-scale comprehensive analysis of internal structures of fossil samples according to claim 6, wherein the number of short scale protrusions is the same as the number of long scale protrusions, the short scale protrusions and the long scale protrusions are in one-to-one correspondence, and the short scale protrusions and the long scale protrusions are vertically intersected at right angles;

or the number of the short scale protrusions is larger than that of the long scale protrusions, and each long scale protrusion perpendicularly intersects with a corresponding short scale protrusion at a right angle.

8. The method of claim 6, wherein step D comprises the steps of:

step D1: scanning the embedded block again by using 3D-XRM at the resolution of more than 10 mu m for 25+/-5 minutes to obtain low-resolution 3D-XRM data;

step D2: and C, sequentially importing the low-resolution 3D-XRM data and the high-resolution 3D-XRM data obtained in the step B into three-dimensional image processing software, aligning the two groups of data, and acquiring the intersection line of the target to-be-cut surface of the embedding block and the surface of the sample, so as to determine the relative position of the region of interest inside the fossil sample and the scale on the surface of the embedding block.

9. The method of claim 1, wherein in step E, scribing is performed on the surface of the embedded block using either of the following two schemes:

the first scheme is as follows: using a diamond scribing pen to scribe on two adjacent surfaces of the embedding block to obtain the intersection line;

the second scheme is as follows: laser etching is performed on two adjacent surfaces of the embedding block by using a 193nm excimer laser ablation system, so as to obtain the intersection line.

10. The method for cross-scale integrated analysis of internal structures of fossil samples according to claim 1, wherein in step F, the ROI is further exposed or polished by using a focused ion beam carried by FIB-SEM and Atlas5 software, and the method specifically comprises the following steps:

step F1: adhering the sheet containing the ROI on a 45-degree pre-inclined aluminum nail table;

step F2: placing the aluminum nail table with the adhered sheet into a coating instrument, coating the film, and then placing the coated aluminum nail table into an FIB-SEM;

step F3: tilting the sample holder/aluminum nail table by 45 degrees to enable the sample holder/aluminum nail table to be parallel to the ion beam machining direction, and shooting SEM images; if the ROI is exposed, performing the direct step F6; if the surface is close to the ROI area, sequentially performing the steps F4-F6;

Step F4: the sample surface SEM image is initially aligned with the 3D-XRM image;

step F5: ion beam cutting is carried out on the position corresponding to the internal ROI; step F6: the SEM data exposing the ROI are realigned with the 3D-XRM data to ultimately determine the three-dimensional reconstruction region of the sample.