Method and device for correlated imaging of biological sample

Abstract

The invention provides a method and a device for correlated imaging of a biological sample, and the method comprises the following steps: freezing and fixing the biological sample, and directly converting water in cells or tissues of the biological sample into a glassy state to obtain a frozen biological sample; performing electron microscope imaging on the frozen biological sample, and determining a to-be-cut position of the frozen biological sample; the optical signal intensity corresponding to the to-be-cut position meets a preset condition; optical imaging is conducted on the to-be-cut position, three-dimensional optical image data are obtained, and the three-dimensional optical image data comprise specific optical information of the to-be-cut position; performing continuous cutting on the to-be-cut position, and performing two-dimensional electron microscope imaging on a cut exposed surface to obtain a plurality of electron microscope images; and reconstructing the plurality of electron microscope images into a three-dimensional electron microscope image, and carrying out correlated imaging on the three-dimensional optical image data and the three-dimensional electron microscope image to obtain the three-dimensional ultrastructure of the biological sample.

Description

Technical Field

[0001] This disclosure relates to the field of life science technology, specifically to the field of correlation imaging technology, and in particular to a method and apparatus for correlation imaging of biological samples. Background Technology

[0002] With the continuous development of the life sciences, obtaining high-resolution three-dimensional structural information of cells and tissues has become a key approach and urgent need for exploring the mysteries of life. Volume electron microscopy (vEM), as an emerging imaging technology, can resolve the three-dimensional ultrastructure of samples at nanometer resolution. vEM overcomes the limitations of traditional electron microscopy, which can only obtain two-dimensional images, and through innovative methods of continuous slicing and sequential imaging, has successfully achieved complete reconstruction of the high-resolution three-dimensional structure of large-scale samples. In recent years, vEM technology has entered a stage of rapid development, significantly promoting the advancement of cell biology, neuroscience, medicine, and other fields.

[0003] Based on the imaging method, vEM technology can be divided into two main categories: vEM based on transmission electron microscopy (TEM) and vEM based on scanning electron microscopy (SEM). Among these, VEM based on SEM has become the dominant force in vEM due to its simple workflow, high experimental throughput, and wide range of applications. However, vEM technology also faces a series of challenges. In terms of sample preparation, traditional chemical fixation or cryopreservation methods involve multiple chemical treatment steps, such as aldehyde cross-linking, organic dehydration, and heavy metal staining. These steps can significantly impact the ultrastructure of cells, easily leading to sample distortion and artifacts. Summary of the Invention

[0004] In view of the above problems, this disclosure provides a method and apparatus for correlation imaging of biological samples, which at least partially solves the above technical problems.

[0005] This disclosure provides a method for correlated imaging of biological samples, comprising: cryo-fixing the biological sample to directly convert water in the cells or tissues of the biological sample into a glassy state, thereby obtaining a frozen biological sample; performing electron microscopy imaging on the frozen biological sample to determine the cutting position of the frozen biological sample; ensuring that the optical signal intensity corresponding to the cutting position meets preset conditions; performing optical imaging on the cutting position to obtain three-dimensional optical image data, the three-dimensional optical image data including specific optical information of the cutting position; performing continuous cutting on the cutting position and performing two-dimensional electron microscopy imaging on the exposed cutting surface to obtain multiple electron microscopy images; reconstructing the multiple electron microscopy images into a three-dimensional electron microscopy image, and correlated imaging of the three-dimensional optical image data with the three-dimensional electron microscopy image to obtain the three-dimensional ultrastructure of the biological sample.

[0006] According to embodiments of this disclosure, freezing and fixing biological samples to directly convert water in the cells or tissues of the biological sample into a glassy state to obtain frozen biological samples includes: uniformly distributing the biological sample on a specially designed carrier; rapidly immersing the carrier carrying the biological sample into a liquid coolant, and using ultra-fast cooling to directly vitrify the water to obtain frozen biological samples.

[0007] According to embodiments of this disclosure, freezing and fixing biological samples to directly convert the water in the cells or tissues of the biological samples into a glassy state, thereby obtaining frozen biological samples, further includes: placing the biological samples in a high-pressure environment, adding liquid nitrogen to cool them to a low temperature, and under the synergistic effect of high pressure and low temperature, directly converting the water in the cells or tissues of the biological samples into a glassy state, thereby obtaining frozen biological samples.

[0008] According to embodiments of this disclosure, electron microscopy imaging of frozen biological samples to determine the cutting location of the frozen biological samples includes: scanning the frozen biological samples at low magnification using an electron beam or ion beam to identify target areas with intact morphology and uniform ice layers; observing the morphology, ice layer thickness, and boundary clarity of the target areas using an electron beam or ion beam at high magnification to determine candidate locations; performing fluorescence imaging on the candidate locations, and determining the candidate locations with fluorescence signals higher than a preset value as the cutting locations based on the fluorescence imaging results.

[0009] According to embodiments of this disclosure, before optical imaging of the cut location, the method further includes: depositing a multilayer film structure consisting of a conductive layer, a protective layer, and another conductive layer sequentially stacked on the surface of a frozen biological sample; the conductive layer is used to increase the conductivity of the frozen biological sample to improve the electronic imaging contrast and reduce the charge accumulation effect; the protective layer is used to protect the surface of the frozen biological sample from being cut during the cutting process, while also reducing the curtain effect generated by the cutting.

[0010] According to an embodiment of this disclosure, continuous cutting of the position to be cut includes: during the cutting process, drift correction of the position of each cut based on a first mark pre-added to the cutting area.

[0011] According to embodiments of this disclosure, a two-dimensional electron microscope (TEM) image is performed on the cut exposed surface to obtain multiple TEM images, including: during the TEM imaging process, charge removal is performed on the surface of the frozen biological sample, and the charge removal method includes at least one of image averaging, gas-assisted charge neutralization, and electron beam scanning pattern optimization.

[0012] According to embodiments of this disclosure, the method further includes: adding a second marker to the location to be cut; associating three-dimensional optical image data with three-dimensional electron microscope images for imaging, including: registering the three-dimensional optical image data and the three-dimensional electron microscope images based on the second marker to perform associative imaging.

[0013] According to embodiments of this disclosure, associating three-dimensional optical image data with three-dimensional electron microscope images for imaging further includes: registering the three-dimensional optical image data and the three-dimensional electron microscope images based on common cell structures.

[0014] Another aspect of this disclosure provides an apparatus for correlated imaging of biological samples, comprising: a freezing module for freezing and fixing biological samples, directly converting water in the cells or tissues of the biological sample into a glassy state to obtain a frozen biological sample; a determination module for performing electron microscopy imaging on the frozen biological sample to determine the cutting position of the frozen biological sample, wherein the optical signal intensity corresponding to the cutting position meets preset conditions; an optical imaging module for performing optical imaging on the cutting position to obtain three-dimensional optical image data, the three-dimensional optical image data including specific optical information of the cutting position; an electron microscopy image imaging module for continuously cutting the cutting position and performing two-dimensional electron microscopy imaging on the exposed cutting surface to obtain multiple electron microscopy images; and a correlated imaging module for reconstructing the multiple electron microscopy images into a three-dimensional electron microscopy image and correlated imaging of the three-dimensional optical image data with the three-dimensional electron microscopy image to obtain the three-dimensional ultrastructure of the biological sample.

[0015] The method and apparatus for correlation imaging of biological samples provided in this disclosure have at least the following technical effects:

[0016] By cryo-fixing biological samples, the water in the cells or tissues of the biological samples is directly converted into a glassy state, which can completely preserve the fluorescent label and make photoelectric correlation imaging possible. It enables multimodal characterization of biological samples by cryo-light microscopy and cryo-electron microscopy, and organically combines the specificity of fluorescence imaging with the nanoscale resolution of electron microscopy imaging, achieving a triple improvement in efficiency, fidelity and imaging modality.

[0017] Rapid cryofixation not only significantly simplifies the sample preparation process, avoids complex chemical fixation or cryofixation alternatives, and significantly improves sample preparation efficiency, but also effectively prevents damage to cell structures during sample preparation, enabling true imaging of cells in their natural physiological state.

[0018] During the cutting process, the pre-set marks in the cutting area are tracked and positioned, and the cutting position is corrected in real time to ensure the uniformity and continuity of the cutting thickness, thus improving the quality of the electron microscope image.

[0019] Before cutting, a multilayer film structure is formed by evaporating a conductive layer, a protective layer, and another conductive layer. The conductive layer increases the conductivity of the sample, improves the contrast of electronic imaging, and reduces the charge accumulation effect. The protective layer protects the sample surface from being cut during the focused ion beam cutting process and can also reduce the curtain effect caused by cutting, thereby improving the imaging quality.

[0020] Using charge removal methods to remove charge from the surface of frozen biological samples can reduce the charge accumulation effect generated during secondary electron imaging, thereby improving image contrast and resolution. Attached Figure Description

[0021] The foregoing contents, as well as other objects, features, and advantages of this disclosure, will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0022] Figure 1 A flowchart illustrating a method for correlated imaging of biological samples according to an embodiment of the present disclosure is shown schematically.

[0023] Figure 2 A schematic diagram illustrating the results of fluorescence imaging according to an embodiment of the present disclosure is shown.

[0024] Figure 3 A schematic diagram of a first mark according to an embodiment of the present disclosure is shown.

[0025] Figure 4 The illustration shows a cell cryogenic secondary electron imaging result according to an embodiment of the present disclosure.

[0026] Figure 5 A schematic diagram of a three-dimensional electron microscope image according to an embodiment of the present disclosure is shown.

[0027] Figure 6 A schematic diagram of a second mark according to an embodiment of the present disclosure is shown.

[0028] Figure 7 This is a block diagram of an apparatus for correlated imaging of biological samples according to embodiments of the present disclosure. Detailed Implementation

[0029] The embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the disclosure. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present disclosure for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure.

[0030] Figure 1 A flowchart illustrating a method for correlated imaging of biological samples according to an embodiment of the present disclosure is shown schematically.

[0031] like Figure 1 As shown, the method for correlated imaging of biological samples in this embodiment may include operations S110 to S150.

[0032] In operation S110, biological samples are cryo-fixed, which directly converts the water in the cells or tissues of the biological sample into a glassy state, resulting in frozen biological samples.

[0033] During operation S120, electron microscopy imaging is performed on the frozen biological sample to determine the location to be cut.

[0034] In operation S130, optical imaging is performed on the position to be cut to obtain three-dimensional optical image data, which includes specific optical information of the position to be cut.

[0035] During operation S140, continuous cutting is performed on the cutting position, and two-dimensional electron microscopy imaging is performed on the exposed cutting surface to obtain multiple electron microscopy images.

[0036] In operation S150, multiple electron microscope images are reconstructed into three-dimensional electron microscope images, and the three-dimensional optical image data is correlated with the three-dimensional electron microscope images to obtain the three-dimensional ultrastructure of the biological sample.

[0037] According to embodiments of this disclosure, cryofixation directly transforms water in the cells or tissues of biological samples into a glassy state through rapid freezing (capable of completion within milliseconds). This ensures the simultaneous fixation of all macromolecular components, converting water from a liquid to an amorphous (glassy) state without the formation of ice crystals. This minimizes damage to the ultrastructure of the biological sample and faithfully preserves the cell structure. This method preserves the original structure and functional state of biological samples, avoiding deformation and component loss caused by traditional chemical fixation or drying processes, thus providing a reliable sample basis for high-resolution imaging.

[0038] According to embodiments of this disclosure, the site to be cut can be a cell in a frozen biological sample whose optical signal intensity meets preset conditions. Meeting preset conditions can be understood as the optical properties (such as fluorescence intensity, scattered light intensity, etc.) of the site to be cut must reach the threshold of the experimental design to ensure that the target molecule, structure, or function can be clearly and accurately detected, located, or quantified by optical microscopy or other imaging techniques.

[0039] According to embodiments of this disclosure, the specific optical information of the location to be cut can refer to the optical features that can uniquely identify the target cell or its specific internal structure / molecule, obtained through optical imaging techniques (such as fluorescence microscopy, confocal microscopy, light sheet microscopy, etc.).

[0040] According to embodiments of this disclosure, two-dimensional electron microscopy imaging can be performed on the exposed cutting surface after each cutting of the position to be cut.

[0041] In some embodiments, freezing and fixing biological samples to directly convert the water in the cells or tissues of the biological samples into a glassy state to obtain frozen biological samples includes: uniformly distributing the biological samples on a specially designed carrier; rapidly immersing the carrier carrying the biological samples into a liquid coolant, and using ultra-rapid cooling to directly vitrify the water to obtain frozen biological samples.

[0042] For example, the ultrafast cooling rate can be greater than or equal to 10. 5 K / s. Coolants include, but are not limited to, ethane and propane. A specially designed support grid is made of copper or molybdenum and covered with a porous carbon or gold film to support the sample and control the ice thickness.

[0043] In other embodiments, freezing and fixing biological samples to directly convert water in the cells or tissues of the biological sample into a glassy state to obtain frozen biological samples may further include: placing the biological sample in a high-pressure environment and adding liquid nitrogen to cool it to a low temperature, so that the water in the cells or tissues of the biological sample is directly converted into a glassy state under the synergistic effect of high pressure and low temperature to obtain frozen biological samples.

[0044] For example, the pressure of the high-pressure environment can be determined based on the water content of the biological sample. For instance, a soft tissue sample with a water content of 70% can be non-destructively frozen to a depth of 200 μm at maximum pressure (2000 bar). In one example of this disclosure, the sample was placed in a high-pressure environment of approximately 2100 bar and then instantly cooled to liquid nitrogen temperature.

[0045] Electron microscopy imaging of frozen biological samples to determine the cutting location can include: scanning the frozen biological sample at low magnification using an electron beam or ion beam to identify target areas with intact morphology and uniform ice layer; observing the morphology, ice layer thickness, and boundary clarity of the target area using an electron beam or ion beam at high magnification to determine candidate locations; performing fluorescence imaging on the candidate locations, and determining the candidate locations with fluorescence signals higher than a preset value as the cutting locations based on the fluorescence imaging results.

[0046] Figure 2 A schematic diagram illustrating the results of fluorescence imaging according to an embodiment of the present disclosure is shown.

[0047] like Figure 2 As shown, for the currently identified candidate locations, fluorescence imaging can be used to determine whether the cell fluorescent labeling is sufficient. If the fluorescence signal is not obvious or cannot be detected, the process can be repeated: use an electron beam or ion beam to scan the frozen biological sample at low magnification to identify the target area with intact morphology and uniform ice layer; use an electron beam or ion beam at high magnification to observe the morphology, ice layer thickness, and boundary clarity of the target area, and re-determine another candidate location. Then, use fluorescence imaging again to determine whether the cell fluorescent labeling is sufficient until the fluorescence signal of the identified candidate location is obvious. Once the candidate location is determined, it is designated as the location to be cut, and a set of three-dimensional optical image data is acquired.

[0048] For example, the low magnification can be 20x to 2000x, and the high magnification can be 1000x to 800000x, but this disclosure does not limit the specific magnification.

[0049] In some embodiments, before optical imaging of the cut location, the method may further include: depositing a multilayer film structure consisting of a conductive layer, a protective layer, and another conductive layer sequentially stacked on the surface of the frozen biological sample; the conductive layer is used to increase the conductivity of the frozen biological sample to improve the electronic imaging contrast and reduce the charge accumulation effect; the protective layer is used to protect the surface of the frozen biological sample from being cut during the cutting process, while also reducing the curtain effect generated by the cutting.

[0050] For example, a sample is coated with a metallic platinum (Pt) conductive layer (sputter coating), then a compound platinum protective layer is coated using the gas injection system (GIS) of an electron microscope, and then a metallic platinum (Pt) conductive layer is coated again to form a triple coating structure of conductive-protective-conductive.

[0051] In some embodiments, continuous cutting of the position to be cut may include: during the cutting process, drift correction of the position of each cut based on a first mark pre-added to the cutting area.

[0052] Figure 3 A schematic diagram of a first mark according to an embodiment of the present disclosure is shown.

[0053] like Figure 3 As shown, a cross mark can be placed near the cutting area. Before each cut, the mark is tracked and positioned, and the cutting position is adjusted in real time to ensure uniformity and continuity of the cutting thickness.

[0054] According to embodiments of this disclosure, a focused ion beam can be used to continuously cut cells and perform secondary electron imaging on each exposed surface until the observed volume is completely imaged.

[0055] Figure 4 The illustration shows a cell cryogenic secondary electron imaging result according to an embodiment of the present disclosure.

[0056] from Figure 4 As can be seen, after cryo-fixation, the electron imaging is clear, and high-quality electron microscope images are obtained.

[0057] Figure 5 A schematic diagram of a three-dimensional electron microscope image according to an embodiment of the present disclosure is shown.

[0058] like Figure 5 As shown, by reconstructing two-dimensional data continuously acquired by a scanning electron microscope into a three-dimensional image, it is possible to obtain complete three-dimensional electron microscope image information of the imaging volume block.

[0059] In some embodiments, performing two-dimensional electron microscopy imaging on the cut exposed surface to obtain multiple electron microscopy images may include: during the two-dimensional electron microscopy imaging process, performing charge removal on the surface of the frozen biological sample, wherein the charge removal method includes at least one of image averaging processing method, gas-assisted charge neutralization method, and electron beam scanning pattern optimization method.

[0060] For example, image averaging methods may include line averaging, frame averaging, and other image averaging methods. These methods reduce noise and indirectly suppress charge effects through signal processing, making them suitable for preliminary observation of samples with low conductivity. Line averaging can average multiple sampled signals from the same scan line, reducing random noise interference. Frame averaging can perform pixel-level averaging on multiple consecutive frames of images, eliminating random noise and transient charge effects.

[0061] Gas-assisted charge neutralization can neutralize accumulated charges by introducing trace amounts of gas or charged ions onto the sample surface, making it suitable for real-time processing of non-conductive samples. Neutral gases (such as nitrogen or water vapor) can be sprayed onto the sample surface; the gas molecules adsorb the charge and form a conductive layer. Charged ions (such as Ar) can also be sprayed. + O2 -It neutralizes the charge on the sample surface, forming an electrically neutral layer.

[0062] Electron beam scanning pattern optimization can reduce charge accumulation hotspots by controlling the electron beam scanning path, making it suitable for high-resolution imaging and processing. For example, scanning along a serpentine path avoids repeatedly irradiating the same area, reducing local charge accumulation. Another example is randomly jumping the electron beam to different locations to break the regularity of charge accumulation.

[0063] In some embodiments, the method may further include adding a second mark to the location to be cut.

[0064] Figure 6 A schematic diagram of a second mark according to an embodiment of the present disclosure is shown.

[0065] like Figure 6 As shown, after electron beam / ion beam imaging of the sample and searching for candidate locations based on features such as cell morphology and ice thickness, a target (e.g., a cross) can be applied around the candidate location (e.g., on the crossbeam of the electron microscope grid) using a focused ion beam (FIB) to serve as a reference for subsequent photoelectric correlation registration.

[0066] Based on this, associating three-dimensional optical image data with three-dimensional electron microscope images for imaging can include: matching three-dimensional optical image data with three-dimensional electron microscope images based on a second marker to perform associative imaging.

[0067] In other embodiments, associating three-dimensional optical image data with three-dimensional electron microscope images may further include: registering based on common cellular structures in the three-dimensional optical image data and the three-dimensional electron microscope images.

[0068] It should be understood that for high-precision, multi-scale, or dynamic imaging, and where the sample allows the introduction of markers (such as fixed cells or in vitro cultured cells), registration can be performed based on the added markers. For scenarios where marker interference needs to be avoided and the sample structure is clear and stable (such as fixed tissue sections or high-contrast electron microscopy images), registration can be performed based on the cell structures common to the three-dimensional optical image data and the three-dimensional electron microscopy image.

[0069] The following will combine Figure 7 An apparatus for correlated imaging of biological samples according to one embodiment of the present disclosure is illustrated below. Figure 7 This is a block diagram of an apparatus for correlated imaging of biological samples according to embodiments of the present disclosure.

[0070] like Figure 7As shown, the device 700 may include a cryo-module 710, a determination module 720, an optical imaging module 730, an electron microscope image imaging module 740, and a correlation imaging module 750.

[0071] The freezing module 710 is used to freeze-fix biological samples, directly converting the water in the cells or tissues of the biological sample into a glassy state to obtain a frozen biological sample. In one embodiment, the freezing module 710 can be used to perform the operation S110 described above, which will not be repeated here.

[0072] The determination module 720 is used to perform electron microscopy imaging on the frozen biological sample to determine the location to be cut; the optical signal intensity corresponding to the location to be cut meets preset conditions. In one embodiment, the determination module 720 can be used to perform the operation S120 described above, which will not be repeated here.

[0073] The optical imaging module 730 is used to perform optical imaging on the location to be cut, obtaining three-dimensional optical image data, which includes specific optical information of the location to be cut. In one embodiment, the optical imaging module 730 can be used to perform the operation S130 described above, which will not be repeated here.

[0074] The electron microscope image imaging module 740 is used to continuously cut the area to be cut and perform two-dimensional electron microscope imaging on the exposed cutting surface to obtain multiple electron microscope images. In one embodiment, the electron microscope image imaging module 740 can be used to perform the operation S140 described above, which will not be repeated here.

[0075] The associated imaging module 750 is used to reconstruct multiple electron microscope images into a three-dimensional electron microscope image, and to correlate the three-dimensional optical image data with the three-dimensional electron microscope image to obtain the three-dimensional ultrastructure of the biological sample. In one embodiment, the associated imaging module 750 can be used to perform the operation S150 described above, which will not be repeated here.

[0076] It should be noted that the specific implementation details in the device embodiment section are the same as or similar to the specific details in the method embodiment section. Please refer to the device embodiment section for details, which will not be repeated here.

[0077] The embodiments of this disclosure have been described above. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. Although various embodiments have been described above, this does not mean that the measures in the various embodiments cannot be used advantageously in combination. The scope of this disclosure is defined by the appended claims and their equivalents. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of this disclosure, and all such substitutions and modifications should fall within the scope of this disclosure.

Claims

1. A method for correlation imaging of biological samples, characterized in that, include: Cryofixation of biological samples allows water in the cells or tissues of the biological sample to be directly converted into a glassy state, resulting in frozen biological samples. The frozen biological sample was imaged using an electron microscope to determine the location to be cut; the optical signal intensity corresponding to the location to be cut met a preset condition. Optical imaging is performed on the location to be cut to obtain three-dimensional optical image data, which includes specific optical information of the location to be cut. The cutting position is continuously cut, and the exposed cutting surface is imaged by two-dimensional electron microscopy to obtain multiple electron microscopy images; Multiple electron microscopy images are reconstructed into three-dimensional electron microscopy images, and the three-dimensional optical image data is correlated with the three-dimensional electron microscopy images to obtain the three-dimensional ultrastructure of the biological sample.

2. The method according to claim 1, characterized in that, The method of cryofixing biological samples, which directly converts the water in the cells or tissues of the biological sample into a glassy state to obtain frozen biological samples, includes: The biological sample is evenly distributed on a specially designed carrier net; the carrier net carrying the biological sample is rapidly immersed in a liquid coolant, and the water is directly vitrified by ultra-fast cooling to obtain the frozen biological sample.

3. The method according to claim 1, characterized in that, The method of cryofixing biological samples, which directly converts the water in the cells or tissues of the biological samples into a glassy state to obtain frozen biological samples, further includes: The biological sample is placed in a high-pressure environment and cooled to a low temperature by adding liquid nitrogen. Under the synergistic effect of high pressure and low temperature, the water in the cells or tissues of the biological sample is directly converted into a glassy state, resulting in a frozen biological sample.

4. The method according to claim 1, characterized in that, The step of performing electron microscopy imaging on the frozen biological sample to determine the location to be cut includes: The frozen biological sample was scanned at low magnification using an electron beam or ion beam to identify target areas in the frozen biological sample that were morphologically intact and had a uniform ice layer. Using an electron beam or ion beam at high magnification, the morphology, ice thickness, and boundary clarity of the target area are observed to determine candidate locations; Fluorescence imaging is performed on the candidate positions, and the candidate positions with fluorescence signals higher than a preset value are determined as the positions to be cut based on the fluorescence imaging results.

5. The method according to any one of claims 1 to 4, characterized in that, Before optical imaging the area to be cut, the method further includes: A multilayer film structure consisting of a conductive layer, a protective layer, and another conductive layer sequentially deposited on the surface of the frozen biological sample. The conductive layer is used to increase the conductivity of the frozen biological sample in order to improve the contrast of electronic imaging and reduce the charge accumulation effect; The protective layer is used to protect the surface of the frozen biological sample from being cut during the cutting process, while also reducing the curtain effect caused by cutting.

6. The method according to claim 1, characterized in that, The continuous cutting of the position to be cut includes: During the cutting process, the position of each cut is corrected for drift based on a first mark pre-added to the cutting area.

7. The method according to claim 1, characterized in that, The two-dimensional electron microscope imaging of the exposed cutting surface yields multiple electron microscope images, including: During two-dimensional electron microscopy imaging, the charge on the surface of the frozen biological sample is eliminated. The charge elimination method includes at least one of image averaging, gas-assisted charge neutralization, and electron beam scanning pattern optimization.

8. The method according to claim 1, characterized in that, The method further includes: Add a second mark to the location to be cut; The step of associating the three-dimensional optical image data with the three-dimensional electron microscope image includes: The three-dimensional optical image data and the three-dimensional electron microscope image are registered based on the second marker to perform correlated imaging.

9. The method according to claim 1 or 8, characterized in that, The step of associating the three-dimensional optical image data with the three-dimensional electron microscope image for imaging further includes: Registration is performed based on the common cell structures in the three-dimensional optical image data and the three-dimensional electron microscopy image.

10. A device for correlated imaging of biological samples, characterized in that, include: The freezing module is used to freeze-fix biological samples, so that the water in the cells or tissues of the biological samples is directly converted into a glassy state to obtain frozen biological samples. A determination module is used to perform electron microscopy imaging on the frozen biological sample to determine the location to be cut in the frozen biological sample; The optical signal intensity at the location to be cut meets the preset condition; An optical imaging module is used to perform optical imaging on the location to be cut to obtain three-dimensional optical image data, wherein the three-dimensional optical image data includes specific optical information of the location to be cut. An electron microscope image imaging module is used to continuously cut the position to be cut and perform two-dimensional electron microscope imaging on the cut exposed surface to obtain multiple electron microscope images; The associated imaging module is used to reconstruct multiple electron microscope images into a three-dimensional electron microscope image, and associate the three-dimensional optical image data with the three-dimensional electron microscope image to obtain the three-dimensional ultrastructure of the biological sample.

Images