Imaging assistance apparatus, imaging method for biological tissue sample, and laser imaging system

Abstract

An imaging assistance apparatus, an imaging method for a biological tissue sample, and a laser imaging system (03). The imaging assistance apparatus comprises: a sample carrying apparatus (01), which carries a biological tissue sample (M); a position measurement apparatus (04), which performs measurement on the sample carrying apparatus (01) in a z-axis direction, and is used for measuring the position of cover glass (101a) at the bottom of the sample carrying apparatus (01) when the biological tissue sample (M) is pressed in the z-axis direction, so as to obtain a position parameter of the biological tissue sample (M) in the z-axis direction, and for controlling, during an imaging process, a laser imaging system (03) to perform rapid focusing on the biological tissue sample (M) in the z-axis direction; or / and a positioning observation device (02), which observes the position of the biological tissue sample (M) in an xy plane, detects position information of the biological tissue sample (M) in the sample carrying apparatus (01) in the xy plane, and controls, during the imaging process, the laser imaging system (03) to perform rapid alignment in the xy plane.

Description

Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system Technical Field

[0001] The present application relates to the field of optical technology, and in particular to an imaging auxiliary device, an imaging method for biological tissue samples, and a laser imaging system. Background Art

[0002] Multiphoton fluorescence technology is widely used for precise imaging of biological tissue samples without damaging the samples. It does not damage the biological tissue samples during the imaging process and can completely preserve all kinds of original biological information in the biological tissue samples. This technology generally places the tissue sample on a sample carrier, which is then placed on a sample stage behind the objective lens of the laser imaging system. The laser is irradiated onto the tissue sample through the objective lens of the optical system, and the characteristic (excitation) fluorescence generated by the laser on the biological tissue sample is collected, thereby completing the imaging process of the biological tissue sample.

[0003] Due to the wide variety and varying sizes of biological tissue samples, the field of view (FOV) of multiphoton precision imaging equipment based on multiphoton fluorescence technology is limited and generally much smaller than the size of the biological tissue sample. Furthermore, changes over time when placed directly on a sample stage are difficult to accurately predict. Consequently, when multiphoton imaging of tissue samples is used in the field, image blurring frequently occurs, locking onto the target z-axis focal plane takes a long time, and rapid determination of the field of view focus within the corresponding xy focal plane is difficult. This is particularly true for smaller biological tissue samples (typically tens to hundreds of cells), such as animal embryos. Because these samples are much smaller than the bottom area of ​​the sample carrier, they often require positioning using a microscope before being placed on the imaging device for imaging based on the positioning data. However, due to the cumbersome and time-consuming z-axis focusing of various inverted microscopes, which require a large range for focusing, a significant amount of time is required to locate the sample on the sample carrier during imaging, significantly impacting imaging efficiency.

[0004] Summary of the Invention

[0005] Based on this, it is necessary to provide an imaging auxiliary device, an imaging method for biological tissue samples and a laser imaging system to address at least one of the above technical defects, so as to improve the imaging operation efficiency and imaging effect.

[0006] An imaging auxiliary device comprises: a sample carrying device for placing a biological tissue sample to be detected and a positioning observation device for observing the position of the biological tissue sample;

[0007] The sample carrying device is used to carry biological tissue samples;

[0008] The positioning observation device is used to observe the biological tissue sample on the sample carrier before imaging, and to determine the position information of the biological tissue sample on the xy plane in the sample carrier;

[0009] The position information is used to control the objective lens of the laser imaging system to quickly align with the biological tissue sample during the imaging process.

[0010] In one embodiment, a scale is provided on the horizontal surface of the sample holding device to indicate the position information of the target object on the horizontal surface;

[0011] The ruler diagram includes: a coordinate ruler or grid with spatial visual reference scale;

[0012] The positioning observation device determines the position information of the biological tissue sample on the xy plane in the sample carrying device according to the ruler diagram.

[0013] In one embodiment, the positioning observation device includes an optical microscope, which is used to search for the biological tissue sample through an eyepiece and determine the position information of the biological tissue sample in the sample holding device according to the ruler diagram.

[0014] In one embodiment, the positioning observation device includes: a camera and a control module connected thereto;

[0015] The camera is used to take pictures on the horizontal surface of the sample supporting device;

[0016] The control module is used to analyze the image to determine the position information of the biological tissue sample in the sample carrying device.

[0017] In one embodiment, the sample carrying device includes: a sample box, a base, a fixed cover, and a ruler assembly;

[0018] The sample box includes a culture dish and a pressure cover, the scale pattern is provided on the scale assembly, and the scale assembly is connected to the bottom of the base;

[0019] Wherein, the culture dish is used to carry biological tissue samples;

[0020] The structure of the gland matches the culture dish and is used to cover the culture dish;

[0021] The base is used to place the sample box;

[0022] The fixing cover is used to fix the sample box on the base.

[0023] In one embodiment, the scale assembly is designed as a groove, and the base is mounted on the scale assembly in an embedded manner;

[0024] A positioning bead is provided on the side of the base, and a positioning hole is provided on the side of the ruler assembly; wherein the positioning bead and the positioning hole are matched and positioned.

[0025] The technical solution of the above embodiment is to place the biological tissue sample to be tested on a sample carrier device provided with a scale pattern. Before imaging, the position of the biological tissue sample is observed using a positioning observation device to obtain its position information on the horizontal plane of the sample carrier device; this position information is used to control the objective lens of the laser imaging system to quickly align with the biological tissue sample during the imaging process, thereby achieving rapid imaging of the laser imaging system and improving imaging efficiency.

[0026] An imaging auxiliary device comprises: a sample carrying device for placing a biological tissue sample to be detected and a position measuring device for measuring the position of the sample carrying device in the z-axis direction;

[0027] The sample carrying device is used to place the biological tissue sample and compress it in the z-axis direction;

[0028] The position measuring device is used to measure the position of the cover glass at the bottom of the sample carrying device to obtain the position parameters of the biological tissue sample placed on the sample carrying device in the z-axis direction;

[0029] The position parameter is used to control the laser imaging system to quickly focus on the biological tissue sample in the z-axis direction during the imaging process.

[0030] In one embodiment, the position measuring device is further used to measure the deformation of the cover glass at the bottom of the sample holding device in the z-axis direction in real time during the process of compacting the biological tissue sample;

[0031] The deformation amount is used to indicate that the biological tissue sample is compressed and placed at or near a designed focal position in the z-axis direction.

[0032] In one embodiment, the imaging assisting device further includes: a driving device connected to the pressure cover of the sample carrier, for obtaining the deformation of the cover glass in real time during the process of pressing the biological tissue sample, and driving the pressure cover of the sample carrier to press the biological tissue sample according to the deformation, so that the biological tissue sample is placed at or near the designed focal position in the z-axis direction.

[0033] In one embodiment, the position measurement device includes: a light transmitter and a light receiver mounted on a stage, and a measurement control system connected to the light transmitter and the light receiver, respectively;

[0034] The light emitter is used to emit a test light beam toward the cover glass at the bottom of the sample holding device;

[0035] The light receiver is used to receive the reflected light beam of the test light beam reflected by the cover glass at the bottom of the sample supporting device;

[0036] The measurement control system is used to calculate the focal position of the biological tissue sample in the z-axis direction according to the reflected light beam.

[0037] In one embodiment, the light receiver is used to receive a plurality of reflected light beams reflected by the test light beam from a plurality of surfaces of the sample supporting device;

[0038] The measurement control system is also used to calculate the position coordinates of the cover glass surface of the sample supporting device in the z-axis direction in real time based on the multiple reflected light beams, and determine the deformation amount of the cover glass based on the position coordinates.

[0039] In one embodiment, the light emitter is used to emit a test light beam of set wavelength and brightness;

[0040] The light receiver is used to simultaneously receive the reflected light beams reflected by the lower surface of the cover glass, the upper surface of the cover glass, the surface of the biological tissue sample and the pressing surface.

[0041] In one embodiment, the sample carrying device includes: a sample box, a base, and a fixed cover;

[0042] The sample box includes a culture dish and a pressure cover; wherein the bottom of the culture dish is a cover glass for carrying biological tissue samples;

[0043] The structure of the gland matches the culture dish and is used to cover the culture dish;

[0044] The base is used to place the sample box;

[0045] The fixing cover is used to fix the sample box on the base.

[0046] The technical solution of the above embodiment includes a sample carrier and a position measurement device for measuring the sample carrier in the z-axis direction. When the sample carrier is pressed against the biological tissue sample in the z-axis direction, the position measurement device measures the position of the cover glass at the bottom of the sample carrier to obtain the position parameters of the biological tissue sample in the z-axis direction. This position parameter is then used to control the laser imaging system to quickly focus on the biological tissue sample in the z-axis direction during imaging. This technical solution facilitates rapid focusing of the biological tissue sample, enabling rapid imaging of the laser imaging system and improving imaging efficiency.

[0047] Moreover, by measuring the position parameters in real time through the position measurement device, the deformation of the cover glass at the bottom of the sample holding device in the z-axis direction can be obtained in real time during the process of compacting the biological tissue sample, thereby indicating the compaction of the biological tissue sample, so that it can be placed more accurately at the focal position in the z-axis direction, greatly improving the imaging effect.

[0048] An imaging assisting device comprises: a sample carrier for placing a biological tissue sample to be detected, a positioning observation device for observing the position of the biological tissue sample on the xy plane, and a position measuring device for measuring the position of the sample carrier in the z-axis direction;

[0049] The sample carrying device is used to place the biological tissue sample and compress it in the z-axis direction;

[0050] The positioning observation device is used to observe the sample carrying device before imaging to determine the position information of the biological tissue sample on the xy plane in the sample carrying device;

[0051] The position measuring device is used to measure the position of the cover glass at the bottom of the sample carrying device to obtain the position parameters of the biological tissue sample placed on the sample carrying device in the z-axis direction;

[0052] The position information is used to control the objective lens of the laser imaging system to quickly align with the biological tissue sample during the imaging process; and the position parameter is used to control the laser imaging system to quickly focus on the biological tissue sample in the z-axis direction during the imaging process.

[0053] In one embodiment, a scale is provided on the horizontal surface of the sample holding device to indicate the position information of the target object on the horizontal surface;

[0054] The ruler diagram includes: a coordinate ruler or grid with spatial visual reference scale;

[0055] The positioning observation device determines the position information of the biological tissue sample on the xy plane in the sample carrying device according to the ruler diagram.

[0056] In one embodiment, the positioning observation device includes an optical microscope, which is used to search for the biological tissue sample through an eyepiece and determine the position information of the biological tissue sample in the sample holding device according to the ruler diagram.

[0057] In one embodiment, the positioning observation device includes: a camera for observing the position of the biological tissue sample on the xy plane and a control module;

[0058] The camera is used to take pictures of the biological tissue sample on the xy plane;

[0059] The control module is used to identify the boundary of the biological tissue sample through the image, detect the coordinate range of the biological tissue sample on the xy plane, and input the coordinate range into the laser imaging system during imaging;

[0060] Wherein, the laser imaging system matches its scanning range with the input coordinate range when capturing images.

[0061] In one embodiment, the position measuring device is further used to measure the deformation of the cover glass at the bottom of the sample holding device in the z-axis direction in real time during the process of compacting the biological tissue sample;

[0062] The deformation amount is used to indicate that the biological tissue sample is compressed and placed at or near a designed focal position in the z-axis direction.

[0063] In one embodiment, the imaging assisting device further includes: a driving device connected to the pressure cover of the sample carrier, for obtaining the deformation of the cover glass in real time during the process of pressing the biological tissue sample, and driving the pressure cover of the sample carrier to press the biological tissue sample according to the deformation, so that the biological tissue sample is placed at or near the designed focal position in the z-axis direction.

[0064] In one embodiment, the position measurement device includes: a light transmitter and a light receiver mounted on a stage, and a measurement control system connected to the light transmitter and the light receiver, respectively;

[0065] The light emitter is used to emit a test light beam toward the cover glass at the bottom of the sample holding device;

[0066] The light receiver is used to receive the reflected light beam of the test light beam reflected by the cover glass at the bottom of the sample supporting device;

[0067] The measurement control system is used to calculate the focal position of the biological tissue sample in the z-axis direction according to the reflected light beam.

[0068] In one embodiment, the light receiver is used to receive a plurality of reflected light beams reflected by the test light beam from a plurality of surfaces of the sample supporting device;

[0069] The measurement control system is also used to calculate the position coordinates of the cover glass surface of the sample supporting device in the z-axis direction in real time based on the multiple reflected light beams, and determine the deformation amount of the cover glass based on the position coordinates.

[0070] In one embodiment, the light emitter is used to emit a test light beam of set wavelength and brightness;

[0071] The light receiver is used to simultaneously receive the reflected light beams reflected by the lower surface of the cover glass, the upper surface of the cover glass, the surface of the biological tissue sample and the pressing surface.

[0072] In one embodiment, the sample carrying device includes: a sample box, a base, a fixed cover, and a ruler assembly;

[0073] The sample box includes a culture dish and a pressure cover, the scale pattern is provided on the scale assembly, and the scale assembly is connected to the bottom of the base;

[0074] Wherein, the bottom of the culture dish is a cover glass, which is used to carry biological tissue samples;

[0075] The structure of the gland matches the culture dish and is used to cover the culture dish;

[0076] The base is used to place the sample box;

[0077] The fixing cover is used to fix the sample box on the base.

[0078] In one embodiment, the scale assembly is designed as a groove, and the base is mounted on the scale assembly in an embedded manner;

[0079] A positioning bead is provided on the side of the base, and a positioning hole is provided on the side of the ruler assembly; wherein the positioning bead and the positioning hole are matched and positioned.

[0080] A method for imaging a biological tissue sample, comprising:

[0081] placing a biological tissue sample into a sample holding device;

[0082] Performing an initial observation on the sample holding device to determine a target area of ​​the biological tissue sample in the sample holding device;

[0083] The sample carrying device is placed on the sample stage of the laser imaging system, the objective lens of the laser imaging system is controlled to aim at the target area, and the biological tissue sample is quickly imaged.

[0084] In one embodiment, performing an initial observation on a sample carrier to determine a target area of ​​the biological tissue sample in the sample carrier includes:

[0085] Performing an initial observation on the sample carrying device and determining a target area of ​​the biological tissue sample in the sample carrying device according to the scale diagram;

[0086] Wherein, a scale pattern is provided on the horizontal surface of the sample carrying device.

[0087] In one embodiment, the initial observation of the sample carrier to determine the target area of ​​the biological tissue sample in the sample carrier includes:

[0088] placing the sample holding device on the stage of the optical microscope;

[0089] Searching for biological tissue samples through the eyepiece of an optical microscope;

[0090] Observe the target area of ​​the biological tissue sample corresponding to the ruler map;

[0091] Placing the sample carrier on the sample stage of the laser imaging system, controlling the objective lens of the laser imaging system to align with the target area, and quickly imaging the biological tissue sample, includes:

[0092] The target area is introduced into the control software of the laser imaging system, and the control software is used to guide the objective lens of the laser imaging system to focus on the target area, so as to quickly image the biological tissue sample.

[0093] In one embodiment, the initial observation of the sample carrier to determine the target area of ​​the biological tissue sample in the sample carrier includes:

[0094] placing the sample holding device on the sample stage of the laser imaging system;

[0095] Using a camera installed on the sample stage to take a picture of the bottom plate of the sample carrier, and analyzing the picture to determine the target area of ​​the biological tissue sample in the sample carrier;

[0096] Placing the sample carrier on the sample stage of the laser imaging system, controlling the objective lens of the laser imaging system to align with the target area, and quickly imaging the biological tissue sample includes:

[0097] The target area is introduced into the control software of the laser imaging system, and the control software is used to guide the objective lens of the laser imaging system to focus on the target area, so as to quickly image the biological tissue sample.

[0098] A method for imaging a biological tissue sample, comprising:

[0099] placing a biological tissue sample into a sample holding device;

[0100] The position measuring device measures the position of the cover glass of the sample carrying device pressing the biological tissue sample to obtain the position parameters of the biological tissue sample in the z-axis direction;

[0101] The sample carrying device is placed on the sample stage of the laser imaging system, the laser imaging system is controlled to image the biological tissue sample, and rapid focusing in the z-axis direction is performed according to the position parameters during the imaging process.

[0102] In one embodiment, the method for imaging a biological tissue sample further comprises:

[0103] During the process of compressing the biological tissue sample, the biological tissue sample is compressed by the pressing cover of the sample carrying device with a set amplitude;

[0104] Read the position parameters of the biological tissue sample in the z-axis direction in real time, and wait for a set time until the biological tissue sample returns to a stable shape before performing the next compaction operation;

[0105] The pressing operation and the position parameters of the biological tissue sample in the z-axis direction are cyclically performed until the biological tissue sample is restored to a stable shape and is placed at a focal position in the z-axis direction.

[0106] In one embodiment, the method for imaging a biological tissue sample further comprises:

[0107] Performing an initial observation on the sample holding device and determining a target area of ​​the biological tissue sample in the sample holding device based on a scale diagram provided on a horizontal surface of the sample holding device;

[0108] During the imaging process, the objective lens of the laser imaging system is controlled to align with the target area on the xy plane to quickly image the biological tissue sample;

[0109] In one embodiment, the method for imaging a biological tissue sample further comprises:

[0110] A picture of the biological tissue sample on the xy plane is taken, the boundary of the biological tissue sample is identified based on the picture, the coordinate range of the biological tissue sample on the xy plane is detected, and the coordinate range is input into the laser imaging system during imaging, so that its scanning range is matched with the input coordinate range when the image is collected.

[0111] A laser imaging system comprises: an optical system, a sample stage, and laser imaging equipment; wherein the sample stage is used to place the sample carrying device; and the optical system utilizes the imaging auxiliary device to assist in rapid imaging.

[0112] In one embodiment, during the imaging process, the optical system is used to control the objective lens to quickly align with the biological tissue sample in the sample holding device on the xy plane according to the position information, and irradiate the laser on the biological tissue sample; the laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

[0113] In one embodiment, during the imaging process, the optical system quickly focuses the biological tissue sample in the z-axis direction according to the position parameter and irradiates the laser on the biological tissue sample; the laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

[0114] In one embodiment, during the imaging process, the optical system is used to control the objective lens to quickly align the biological tissue sample in the sample carrier in the xy plane according to the position information, and to quickly focus the biological tissue sample in the z-axis direction according to the position parameter, so as to irradiate the biological tissue sample with the laser;

[0115] The laser imaging device collects fluorescence generated on the biological tissue sample to perform imaging on the biological tissue sample.

[0116] The above-mentioned imaging auxiliary device, imaging method of biological tissue samples and technical solutions of the laser imaging system can assist in controlling the laser imaging system to perform rapid imaging during the imaging process, thereby improving imaging efficiency; and the sample carrying device is suitable for various multi-photon precision imaging equipment and can be used to carry various biological tissue samples, facilitating rapid locking of the position of the biological tissue samples, thereby further improving the efficiency of imaging operations and reducing the risk of contamination of biological tissue samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 is a schematic diagram of the "light slicing" effect of multiphoton fluorescence;

[0118] FIG2 is an exemplary diagram of an imaging assisting device according to an embodiment;

[0119] FIG3 is a schematic diagram of an example xy coordinate ruler;

[0120] FIG4 is a schematic diagram of an example grid;

[0121] FIG5 is a schematic diagram of a positioning observation device according to an embodiment;

[0122] FIG6 is a schematic diagram of a positioning observation device according to another embodiment;

[0123] FIG7 is an exploded view of a sample supporting device according to an embodiment;

[0124] FIG8 is a schematic structural diagram of an exemplary sample carrying device in a separated state;

[0125] FIG9 is a schematic structural diagram of a sample carrying device in an exemplary assembled state;

[0126] FIG10 is a flow chart of a method for imaging a biological tissue sample according to an embodiment;

[0127] FIG11 is an exemplary diagram of an imaging assisting device according to another embodiment;

[0128] FIG12 is a schematic structural diagram of a position measurement device according to an embodiment;

[0129] FIG13 is a flow chart of a method for imaging a biological tissue sample according to another embodiment;

[0130] FIG14 is a flow chart of a method for compressing a biological tissue sample according to an embodiment;

[0131] FIG15 is a flow chart of a method for imaging a biological tissue sample according to another embodiment;

[0132] FIG16 is a schematic diagram of the structure of an exemplary laser imaging system. DETAILED DESCRIPTION

[0133] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0134] It should be noted that due to space limitations, it is impossible to list all technical solutions. Only some implementation plans are provided in the following examples. The "first" and "second" described in this application are only used to distinguish different objects and do not have a substantial distinction.

[0135] During the imaging process, a laser imaging system using multiphoton fluorescence technology generally uses a galvanometer element to achieve imaging of a specific focal plane where the focus is located. As shown in Figure 1, Figure 1 is a schematic diagram of the "light slicing" effect of multiphoton fluorescence. As shown in the figure, the black dot position is the focal position. The imaging mode of the entire biological tissue sample is achieved by cumulative imaging through each focal plane layer by layer, which is called "light slicing" technology imaging. Given the field of view (FOV) size limitation of multiphoton precision imaging equipment, considering that during the imaging process, the objective lens needs to accurately irradiate the laser on the biological tissue sample, and since some biological tissue samples are small in size, it takes a long time to search to locate the biological tissue sample. In order to achieve the positioning of the biological tissue sample, facilitate the rapid imaging acquisition of the biological tissue sample, and improve the imaging operation efficiency, it is not only required to quickly determine the field of view focus in the xy plane, but also to quickly focus the biological tissue sample in the z-axis direction to avoid long-term imaging blur. To this end, the embodiment of the present application provides an imaging auxiliary device that can achieve any of the following functions:

[0136] ① Provide the laser imaging system with accurate position information of the biological tissue sample on the xy plane, so that it can quickly lock the biological tissue sample on the xy plane during the imaging process; assist the laser imaging system in performing rapid imaging.

[0137] ② Provide the position parameters of the coverslip in the z-axis direction for the laser imaging system, so that it can quickly and accurately focus on the biological tissue sample in the z-axis direction during the imaging process; assist the laser imaging system in rapid imaging.

[0138] ③ Simultaneously realize the functions of ① and ② above, and assist the laser imaging system in rapid imaging.

[0139] An embodiment of an imaging assisting device for realizing the above-mentioned function ① is described below.

[0140] Referring to Figure 2, Figure 2 is an example diagram of an imaging auxiliary device according to an embodiment, including: a sample carrying device 01 and a positioning observation device 02; wherein, the sample carrying device 01 places a biological tissue sample M to be detected, and the sample carrying device 01 is provided with a scale pattern on the horizontal plane, which can indicate the position information of the target object on the horizontal plane; the positioning observation device 02 observes the position of the biological tissue sample M, observes the biological tissue sample M on the sample carrying device 01 before imaging, and determines the position information of the biological tissue sample M in the sample carrying device 01 based on the scale pattern; illustratively, by using the scale pattern of the sample carrying device 01, the position information of the biological tissue sample M can be obtained by using the eyes, a magnifying glass, a microscope, etc., so as to identify the boundary contour of the biological tissue sample M. After obtaining the position information, the objective lens 31 of the laser imaging system 03 is assisted in controlling to quickly align with the biological tissue sample M during the imaging process, so that rapid imaging can be performed, thereby improving the imaging efficiency of the laser imaging system 03.

[0141] In one embodiment, the ruler on the sample carrier 01 can be a coordinate ruler or grid with a spatial visual reference scale. As shown in FIG3 , which is an exemplary xy coordinate ruler diagram, the xy coordinate scale can indicate the position of the target object in the sample carrier 01. For example, the shaded area in the figure represents the biological tissue sample M. The coordinate reference scale in the figure can be used to determine the position of the biological tissue sample M in the sample carrier 01. FIG4 , which is an exemplary grid diagram, shows a grid with scales labeled in different order in the vertical and horizontal directions, such as a, b, c, etc. in the horizontal direction and 0, 1, 2, etc. in the vertical direction. The grid can also be distinguished by different colors.

[0142] In one embodiment, as shown in Figure 5, Figure 5 is a schematic diagram of a positioning observation device of an embodiment. The positioning observation device 02 can use an optical microscope 21 to search for the biological tissue sample M through the eyepiece 211, and determine the position information (such as coordinate parameters) of the biological tissue sample M in the sample carrying device 01 according to the scale diagram. Since the optical microscope 21 can observe the tiny biological tissue sample M, the initial positioning of the position of the biological tissue sample M can be completed; specifically, under certain circumstances, the focal position required for imaging (such as the exact center of the biological tissue sample M) can be visually determined, and the corresponding coordinate parameters (x, y) can be recorded.

[0143] After obtaining the position information, it is imported into the control software of the laser imaging system 03. The control software of the laser imaging system 03 pre-stores the scale diagram on the sample carrier 01, and can quickly align the target area according to the position information, thereby assisting in rapid imaging; preferably, an optical microscope 21 with a digital output function can be used, and the optical microscope 21 is connected to the laser imaging system 03 to import the position information of the initially positioned biological tissue sample M into the control software of the laser imaging system 03.

[0144] In another embodiment, as shown in Figure 6, Figure 6 is a schematic diagram of the positioning observation equipment of another embodiment. The positioning observation equipment 02 of this embodiment includes a camera 22 and a control module 23 connected thereto, wherein the camera 22 is arranged above the sample stage 32 of the laser imaging system 03. It should be noted that in a specific implementation, the installation position of the camera 22 can be selected according to different on-site conditions and is not limited to a specific installation position; the camera 22 is used to take pictures on the horizontal plane of the sample supporting device 01; the control module 23 is used to analyze the pictures to determine the position information of the biological tissue sample M in the sample supporting device 01; illustratively, the picture of the biological tissue sample M in the sample supporting device 01 is taken by the camera 22, and the control module 23 can automatically identify the boundary of the biological tissue sample M through the picture, and detect the coordinate range of the biological tissue sample M on the xy plane; during imaging, the coordinate range can be input into the laser imaging system 03, and the laser imaging system 03 can correspond its scanning range on the xy plane to the input coordinate range when taking pictures.

[0145] The control module 23 of this embodiment can be connected to the laser imaging system 03. Before imaging, the sample carrier 01 is placed on the sample table 32, and the control software of the laser imaging system 03 is used to output instructions to the control module 23, and the camera 22 is started to take a picture of the sample carrier 01. By analyzing the picture, the position information (such as coordinate parameters) of the biological tissue sample M in the sample carrier 01 can be determined. The control software of the laser imaging system 03 pre-stores the scale image on the sample carrier 01, and can quickly align with the target area according to the position information, thereby assisting in rapid imaging.

[0146] With respect to the sample carrying device 01 , a specific embodiment thereof will be described below with reference to the accompanying drawings.

[0147] Figure 7 is an exploded view of a sample carrying device 01 according to an embodiment, and Figure 8 is a schematic structural diagram of the sample carrying device 01 in a separated state. As shown in the figure, the sample carrying device 01 according to this embodiment includes: a sample box 10, a ruler assembly 11, a base 12 and a fixed cover 13; wherein, the sample box 10 includes a culture dish 101 and a pressure cover 102, the sample box 10 is made of a transparent material, the culture dish 101 is used to carry the biological tissue sample M, and the structure of the pressure cover 102 matches the culture dish 101; the ruler assembly 11 is connected to the bottom of the base 12, and the ruler assembly 11 can be made of a transparent material; wherein the ruler assembly 11 is marked with a ruler pattern at a hollow position relative to the base 12, and the ruler pattern is used to assist in locating the position of the biological tissue sample M on the culture dish 101.

[0148] During use, the biological tissue sample M to be imaged is first placed in the culture dish 101 and the culture dish 101 is covered; the base 12 is used to place the sample box 10. After the biological tissue sample M is placed, the sample box 10 is placed as a whole on the base 12, and then fixed with the sample box 10 using the fixing cover 13, and finally the base 12 is placed on the ruler assembly 11; after the preparation is completed, the sample carrier 01 is placed on the sample stage 32 for imaging; before imaging, the biological tissue sample M on the sample carrier 01 is scanned by the positioning observation device 02, and the position information of the biological tissue sample M on the culture dish 101 is quickly assisted in positioning according to the ruler diagram; during imaging, the area corresponding to the position parameter is used as the light source focus of the laser imaging system 03, and the objective lens 31 is controlled to quickly determine the field of view focus and lock the focus in the xy focal plane. The laser is irradiated on the biological tissue sample M through the objective lens 31 to collect the fluorescence generated on the biological tissue sample M, and finally the biological tissue sample M is imaged.

[0149] The sample box 10 can be designed as a circular structure, with a circular hollow portion in the middle of the base 12, which is just right for placing the sample box 10. The sample box 10 is then fixed by a fixed cover 13 to ensure stability. Furthermore, the bottom of the culture dish 101 and the bottom of the pressure cover 102 are flat, and the pressure cover 102 is embedded in the culture dish 101 and in close contact. In order to place the biological tissue sample M on the flat bottom surface of the culture dish 101, a sponge pad 103 is also placed at the contact point between the pressure cover 102 and the culture dish 101. The sponge pad 103 can flatten the biological tissue sample M and adhere it to the flat bottom surface of the culture dish 101.

[0150] In this embodiment, the sample cassette flattens the biological tissue sample M against the bottom of the culture dish 101 via the pressing cover 102 and sponge pad 103. This compaction function allows non-standard biological tissue samples M with excessively large z-axis dimensions to be compacted to the z-axis height specified by the sample cassette 10, thereby shortening the time required to lock onto the target z-axis focal plane, better correcting the voids in the scanned image caused by uneven samples, and reducing the frequent occurrence of image blur during the imaging process.

[0151] For the ruler assembly 11, preferably, it can be designed with a groove, and the base 12 is installed on the ruler assembly 11 in an embedded manner; in order to facilitate disassembly and installation and fixation, a positioning bead 121 is provided on the side of the base 12, and a positioning hole 111 is provided on the side of the ruler assembly 11, and the positioning is matched and positioned with the positioning hole 111 by the positioning bead 121; when in use, the base 12 is placed in the groove of the ruler assembly 11 and fixed, and when disassembling, it is only necessary to pull out the base 12.

[0152] In one embodiment, a handle structure 102a is provided on the upper portion of the pressure cap 102. This handle structure 102a assists in pressing the pressure cap 102 onto the bottom surface of the culture dish 101. The sample box 10 can be a disposable component, made of disposable materials, to prevent the biological tissue sample M from coming into contact with other objects, effectively isolate it from the laser imaging system 03, reduce the risk of contamination of the biological tissue sample M, and avoid internal and external contamination, thus complying with relevant requirements of biosafety protection measures. The base 12 and fixed cover 13 are reusable components and can be made of metal. Preferably, the fixed cover 13 is connected to the base 12 via threads. During use, after placing the biological tissue sample M in the sample box 10, the sample box 10 is placed in the hollow part of the base 12. A protrusion can be provided on the sample box 10 and a slot can be provided in the base 12. The position is fixed by inserting the protrusion into the slot, and then the fixing cover 13 is rotated and screwed to push the sample box 10 firmly onto the base 12, thereby avoiding affecting the biological tissue sample M during the movement.

[0153] As in the above embodiment, the base 12 and the fixed cover 13 are reusable and can be used with a disposable sample box to avoid contamination risks while ensuring practicality and effectiveness.

[0154] The sample carrying device 01 provided in the present application is used in a detachable combination, has a compact structure and is easy to operate, as shown in Figure 9, which is a schematic diagram of the structure of the sample carrying device 01 in an exemplary combined state. When in use, the sample carrying device 01 is very compact and the sample box 10 can be quickly replaced; because the sample box 10 is disposable, good isolation and protection of the biological tissue sample M is achieved to prevent contamination, and various types of biological tissue samples M (such as microscopic, puncture biopsy and embryos, etc.) can be accommodated; the base 12, the fixed cover 13 and the ruler assembly 11 are configured for reuse, and the stable base 12 and the fixed cover 13 can place the sample box 10 stably on the sample table 32 to avoid any slight movement during the imaging process.

[0155] The sample carriers provided in the aforementioned embodiments can be used to hold various biological tissue samples. The scale pattern marked on the scale assembly quickly assists in locating the biological tissue sample on the culture dish, assisting the laser imaging system in quickly locking onto the biological tissue sample, thereby improving imaging efficiency. Furthermore, the sample holder of the sample carrier is disposable, preventing the biological tissue sample from coming into contact with other objects, reducing the risk of contamination and complying with relevant biosafety protection measures. The sample holder, using a pressure cap and sponge pad, flattens the biological tissue sample against the bottom of the culture dish, thereby shortening the time required to lock onto the target z-axis focus and reducing the frequent occurrence of image blur during imaging.

[0156] An embodiment of a method for imaging a biological tissue sample is described below.

[0157] Referring to FIG. 10 , FIG. 10 is a flow chart of a method for imaging a biological tissue sample according to an embodiment, comprising the following steps:

[0158] S11, placing the biological tissue sample M into the sample carrying device 01;

[0159] In one embodiment, a scale pattern is provided on the horizontal surface of the sample holding device 01 .

[0160] As shown in Figures 7 to 9, the present application provides an embodiment of a sample carrying device 01, which includes components such as a sample box 10, a scale assembly 11, a base 12, and a fixed cover 13; wherein, the sample box 10 includes a culture dish 101 and a pressure cover 102; wherein, the bottom of the culture dish 101 can be a coverslip.

[0161] Exemplarily, step S11 may include the following:

[0162] s1, firstly, placing the biological tissue sample M to be imaged into the culture dish 101;

[0163] s2, placing a sponge pad 103 on the biological tissue sample M;

[0164] s3, insert the pressure cover 102 to cover the culture dish 101, press the sponge pad 103 to flatten the biological tissue sample M and stick it to the bottom surface of the culture dish 101;

[0165] s4, placing the sample box 10 as a whole on the base 12 and fixing it with the fixing cover 13;

[0166] s5, place the base 12 on the ruler assembly 11 to complete the preparation.

[0167] The ruler diagram may be a coordinate ruler or a grid with a spatial visual reference scale, etc. Of course, other forms of ruler diagrams may also be used to indicate position information.

[0168] S12 , performing an initial observation on the sample carrying device 01 to determine a target area of ​​the biological tissue sample M in the sample carrying device 01 .

[0169] In this step, the positioning observation device 02 can be used to perform initial observation on the sample carrying device 01 and locate the target area of ​​the biological tissue sample M in the sample carrying device 01 according to the scale diagram of the sample carrying device 01.

[0170] In one embodiment, step S12 may include the following:

[0171] The sample carrying device 01 is placed on the stage of the optical microscope 21 , and the biological tissue sample M is searched through the eyepiece 211 of the optical microscope 21 , and the target area of ​​the biological tissue sample M corresponding to the scale image is observed.

[0172] Specifically, the operator can place the sample carrying device 01 on the stage of the optical microscope 21, observe and search for the biological tissue sample M through the eyepiece 211, and at the same time determine the position information corresponding to the biological tissue sample M by observing the scale diagram, such as the coordinate scale, so as to obtain the target area where the biological tissue sample M is located.

[0173] In another embodiment, step S12 may include the following:

[0174] The sample carrier 01 is placed on the sample stage 32 of the laser imaging system 03; a camera 22 installed on the sample stage 32 is used to take a picture of the chassis range of the sample carrier 01, and the picture is analyzed to determine the target area of ​​the biological tissue sample M in the sample carrier 01.

[0175] Specifically, the picture contains the image content of the biological tissue sample M and the ruler image. By analyzing the picture, the image area of ​​the biological tissue sample M can be obtained, and then matched with the ruler image, such as the grid position, the position information of the biological tissue sample M in the sample carrying device 01 can be determined, thereby obtaining the target area where the biological tissue sample M is located.

[0176] S13 , placing the sample carrying device 01 on the sample stage 32 of the laser imaging system 03 , controlling the objective lens 31 of the laser imaging system 03 to align with the target area, and performing rapid imaging of the biological tissue sample M.

[0177] Specifically, according to the target area initially located in step S12, during the process of the laser imaging system 03 imaging the biological tissue sample M, the objective lens 31 can be controlled to quickly align with the target area for imaging.

[0178] In one embodiment, when an optical microscope 21 is used, the corresponding process of step S13 may include: importing the target area into the control software of the laser imaging system 03, guiding the objective lens 31 of the laser imaging system 03 to focus on the target area through the control software, and quickly imaging the biological tissue sample M.

[0179] In addition, if the camera 22 is used to take pictures, the corresponding process of step S13 may include: importing the target area into the control software of the laser imaging system 03, guiding the objective lens 31 of the laser imaging system 03 to focus on the target area through the control software, and quickly imaging the biological tissue sample M.

[0180] An embodiment of an imaging assisting device for realizing the above-mentioned function ② is described below.

[0181] When the biological tissue sample M is pressed tightly by the compaction function, the time for locking the target z-axis focus can be reduced, and the focusing speed in the z-axis direction can be improved. However, due to the differences in the biological tissue samples M and the differences in each placement of the sample carrying device 01, the z-axis focus position of each biological tissue sample M is easily uncontrollable and has a large range of variation. At the same time, since the cover glass 101a at the bottom of the sample carrying device 01 is generally very thin (usually 0.17mm), when the biological tissue sample M is loaded and the pressure cover 102 is pressed on, the cover glass 101a will bend and deform due to the different degrees of compression of the pressure cover 102, which in turn causes the z-axis focus of the biological tissue sample M to change significantly and deviate from the focus position (pre-designed focus position), greatly increasing the difficulty of subsequent z-axis focusing, and in severe cases even leading to focusing failure.

[0182] Accordingly, in order to achieve rapid focusing of the biological tissue sample M in the z-axis direction, as shown in Figure 11, Figure 11 is an example diagram of an imaging auxiliary device according to another embodiment. The imaging auxiliary device of this embodiment includes: a sample carrying device 01 for placing the biological tissue sample M to be detected and a position measuring device 04 for measuring the sample carrying device 01 in the z-axis direction; wherein, the sample carrying device 01 is used to place the biological tissue sample M and press it in the z-axis direction.

[0183] The sample carrier 01 is used to carry a biological tissue sample M and typically includes at least a cover glass 101a and a pressure cover 102. For example, the sample carrier 01 may include a sample box, a base, and a fixed cover. For details, please refer to the relevant content of the aforementioned embodiments and will not be repeated here.

[0184] As for the position measuring device 04, it is used to measure the position of the coverslip 101a at the bottom of the sample carrying device 01, and obtain the position parameters of the biological tissue sample M placed on the sample carrying device 01 in the z-axis direction; the measured position parameters are used to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction during the imaging process; illustratively, when performing position measurement, the position measuring device 04 can perform position measurement in the z-axis direction on multiple positions of the biological tissue sample M within a certain coordinate range on the xy plane, so as to obtain more position parameters, which depends on the specific usage requirements.

[0185] For example, if an optical microscope 21 is used to observe the position of the biological tissue sample M on the xy plane, the position measuring device 04 can be installed under the stage of the optical microscope 21; if a camera 22 located above the sample stage 32 of the laser imaging system 03 is used to take a picture of the biological tissue sample M in the sample carrying device 01 to identify the boundary of the biological tissue sample M and detect the coordinate range of the biological tissue sample M on the xy plane; the position measuring device 04 can be installed under the sample stage 32 of the laser imaging system 03.

[0186] As shown in Figure 11, the cover glass 101a at the bottom of the sample carrier 01 is easily deformed when the biological tissue sample M is pressed; for example, the cover glass 101a can be the bottom of the culture dish 101 of the aforementioned embodiment; at this time, due to the deformation of the cover glass 101a, the biological tissue sample M deviates from the focal position in the z-axis direction. If the coordinate data of the focal position is used during the imaging process, focusing failure will occur. By measuring the position of the cover glass 101a of the sample carrier 01 in the z-axis direction through the position measurement device 04, accurate position parameters, that is, the actual focal position, can be obtained.

[0187] As in the technical solution of the above embodiment, the measured position parameters can be input into the laser imaging system 03 to control its objective lens 31 to quickly focus on the biological tissue sample M in the z-axis direction, thereby significantly improving the imaging operation efficiency and imaging effect.

[0188] In one embodiment, due to the differences between different biological tissue samples M, it is difficult to control the degree of compression each time the biological tissue sample M is placed in the sample carrier 01, so it is impossible to accurately make the biological tissue sample M reach the focal position in the z-axis direction.

[0189] To this end, the imaging assisting device of this embodiment utilizes the position parameters measured in real time by the position measuring device 04 to guide the compression of the biological tissue sample M. Specifically, the position measuring device 04 is further configured to measure the deformation of the cover glass 101a at the bottom of the sample carrier 01 in the z-axis direction in real time during the compression of the biological tissue sample M. This deformation is used to instruct the compression of the biological tissue sample M, placing it at or near the designed focal position in the z-axis direction, as close to the focal position as possible, based on the real-time measurement of the deformation. For example, if the cover 102 is being compressed manually, the deformation of the cover glass 101a in the z-axis direction can be displayed in real time, thereby providing guidance for accurate compression of the biological tissue sample M.

[0190] As in the technical solution of the above embodiment, during measurement, not only the final position parameters of the biological tissue sample M in the z-axis direction are obtained, but also the deformation of the cover glass 101a can be obtained by using the position parameters measured in real time, so as to guide the accurate compression of the biological tissue sample M and avoid excessive compression operation that causes large deformation of the cover glass 101a in the z-axis direction.

[0191] In one embodiment, for the structural scheme of the position measuring device 04, refer to Figure 12. Figure 12 is a schematic structural diagram of the position measuring device of an embodiment, which may specifically include a light emitter 41 and a light receiver 42 installed on the stage, and a measurement control system 43 respectively connected to the light emitter 41 and the light receiver 42; wherein, the light emitter 41 is used to emit a test light beam to the position of the biological tissue sample M at the bottom of the sample carrying device 01 according to the position information of the biological tissue sample M in the sample carrying device 01; the light receiver 42 is used to receive the reflected light beam of the test light beam reflected by the cover glass 101a at the bottom of the sample carrying device 01; illustratively, the light emitter 41 may include a laser head, which emits a laser beam as a test beam under a controlled state, and the light receiver 42 may be a laser probe, which receives the reflected light beam after reflection to realize the laser ranging function.

[0192] The measurement control system 43 may be used to control the light emitter 41 and the light receiver 42 to emit a test light beam and receive a reflected light beam, and calculate the focal position of the biological tissue sample M in the z-axis direction according to the reflected light beam.

[0193] As in the technical solution of the above embodiment, by measuring the changes of the cover glass 101a at the bottom of the sample supporting device 01 by laser ranging, the position parameters of the biological tissue sample M in the z-axis direction can be accurately obtained, which facilitates accurate focusing during the imaging process. At the same time, the deformation of the cover glass 101a can also be obtained in real time to guide the pressing process of the pressure cover 102, so that the biological tissue sample M is placed at or near the designed focal position in the z-axis direction, ensuring that the focal plane of the biological tissue sample M is controllable each time.

[0194] In one embodiment, to more accurately measure the z-axis position parameters of the biological tissue sample M and the deformation of the coverslip 101a, the light emitter 41 can be controlled to emit a test beam with specific parameters, such as a laser beam with a set wavelength and brightness. Simultaneously, the light receiver 42 is configured to receive multiple reflected beams resulting from the test beam reflecting off multiple surfaces of the sample carrier 01, such as the bottom and top surfaces of the coverslip 101a, the surface of the biological tissue sample M, and the surface of the pressure cap 102.

[0195] The measurement control system 43 calculates the position coordinates of the cover glass 101a surface of the sample carrier 01 in the z-axis direction in real time based on the multiple reflected light beams. Since different reflected light beams reflect differently on different targets, the distance to the target surface can be determined by analyzing the optical signals of the reflected light beams. At the same time, the deformation of the cover glass 101a can also be determined based on the measured position coordinates.

[0196] In one embodiment, in order to improve the control accuracy of compacting the biological tissue sample M and enhance the automation efficiency, the imaging auxiliary device of this embodiment may further be provided with a driving device 05 to drive the pressing cover 102 .

[0197] Exemplarily, the driving device 05 can be driven by a motor, and the driving device 05 is connected to the pressure cover 102 of the sample carrier 01. In the process of pressing the biological tissue sample M, the deformation of the cover glass 101a is obtained in real time, and the pressure cover 102 of the sample carrier 01 is driven according to the deformation to press the biological tissue sample M, so that the biological tissue sample M is placed at or near the designed focal position in the z-axis direction.

[0198] As in the technical solution of the above embodiment, the driving device 05 can more accurately drive the pressing cover 102 to press the biological tissue sample M, thereby realizing closed-loop control, so that the biological tissue sample M is closely attached to the cover glass 101a and the deformation of the cover glass 101a is within a controllable range, which facilitates subsequent imaging operations and improves imaging operation efficiency.

[0199] For example, as shown in FIG12 , for the main electrical structure of the imaging auxiliary device, the measurement control system 43 can control the light emitter 41 and the light receiver 42 to perform distance measurement, and is connected to the driving device 05 to control the driving device 05 to press the pressure cover 102 through the real-time measured position data.

[0200] As in the technical solution of the above-mentioned embodiment, by emitting a test light beam with specific parameters and receiving reflected light beams reflected by multiple target surfaces, the distances of different target surfaces are analyzed, so that the position parameters of the biological tissue sample M and the deformation quantities of each component of the sample carrying device 01 can be accurately obtained.

[0201] An embodiment of an imaging assisting device for realizing the above function ③ is described below.

[0202] In one embodiment, the imaging auxiliary device of the present application may include: a sample carrying device 01 for placing the biological tissue sample M to be detected, a position measuring device 04 for measuring the sample carrying device 01 in the z-axis direction, and a positioning observation device for observing the position of the biological tissue sample M on the xy plane.

[0203] The sample carrier 01 can be used to place a biological tissue sample M and compress it in the z-axis direction. A scale is provided on the horizontal surface of the sample carrier 01 to indicate the position information of the target object on the horizontal surface.

[0204] As for the positioning observation equipment, it can be used to observe the biological tissue sample M on the sample carrier 01 before imaging, and determine the position information of the biological tissue sample M on the xy plane in the sample carrier 01 based on the scale diagram; wherein the position information is used to control the objective lens 31 of the laser imaging system 03 to quickly align with the biological tissue sample M during the imaging process.

[0205] As for the position measuring device 04, it can be used to measure the position of the coverslip 101a at the bottom of the sample carrying device 01, and obtain the position parameters of the biological tissue sample M placed on the sample carrying device 01 in the z-axis direction; wherein the position parameters are used to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction during the imaging process.

[0206] For example, the specific structures and functions of the sample carrying device 01, the positioning observation equipment and the position measuring device 04 can be referred to the previous embodiments and will not be repeated here.

[0207] As in the imaging auxiliary device of the above embodiment, the position information of the target area is used to control the objective lens 31 of the laser imaging system 03 to quickly align with the biological tissue sample M in the xy plane during the imaging process, and the position parameters are used to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction, thereby achieving rapid imaging of the laser imaging system 03 and improving imaging efficiency.

[0208] Referring to FIG. 13 , FIG. 13 is a flow chart of a method for imaging a biological tissue sample according to another embodiment, comprising the following steps:

[0209] S21 , placing the biological tissue sample M into the sample carrying device 01 ; the biological tissue sample M may be pressed tightly by the pressing cover 102 .

[0210] S22 , using the position measuring device 04 to measure the position of the cover glass 101 a of the sample carrying device 01 pressing the biological tissue sample M, to obtain position parameters of the biological tissue sample M in the z-axis direction.

[0211] For example, when the sample carrier 01 is placed on the stage of the optical microscope 21 to observe and search for the biological tissue sample M, the position parameters in the z-axis direction are obtained based on the target area of ​​the observed biological tissue sample M using the position measurement device 04.

[0212] S23, placing the sample carrying device 01 on the sample stage 32 of the laser imaging system 03, controlling the laser imaging system 03 to image the biological tissue sample M, and performing rapid focusing in the z-axis direction according to the position parameters during the imaging process.

[0213] For example, the position parameters may be imported into the control software of the laser imaging system 03 , and the control software may be used to guide the objective lens 31 of the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction.

[0214] As in the technical solution of the above embodiment, the position measurement device 04 measures the position of the cover glass 101a to obtain the position parameters of the biological tissue sample M in the z-axis direction, so that when the laser imaging system 03 is imaging, rapid focusing can be performed in the z-axis direction to improve imaging efficiency.

[0215] In one embodiment, in order to more accurately compress the biological tissue sample M in the sample carrier 01, the sample carrier is brought close to the cover glass 101a and positioned at or near the designed focal position in the z-axis direction. The present application further provides a method for compressing the biological tissue sample M, as shown in FIG14 , which is a flow chart of an embodiment of the method for compressing a biological tissue sample, including the following steps:

[0216] s201 , during the process of compressing the biological tissue sample M, the biological tissue sample M is compressed with a set amplitude by the pressing cover 102 of the sample carrying device 01 .

[0217] s202, reading the position parameters of the biological tissue sample M in the z-axis direction in real time to guide the compacting operation.

[0218] s203, stop the pressing operation and wait for a set time (eg, about 5 seconds) until the biological tissue sample M recovers to a stable shape.

[0219] s204, determine whether the biological tissue sample M is placed at (close to) the focal position in the z-axis direction. If so, return to step s202 to perform the next pressing operation; otherwise, execute s205.

[0220] s205, reading the final position parameters of the biological tissue sample M in the z-axis direction, and importing them into the control software of the laser imaging system 03 for imaging.

[0221] As in the technical solution of the above embodiment, the pressing operation is guided by real-time reading of the position parameters of the biological tissue sample M in the z-axis direction, thereby avoiding excessive pressing operation that causes excessive deformation of the cover glass 101a and affects the fast focusing operation of the biological tissue sample M, thereby improving imaging efficiency.

[0222] 15 is a flow chart of a method for imaging a biological tissue sample according to another embodiment, comprising the following steps:

[0223] S31 , placing the biological tissue sample M into the sample carrying device 01 ; wherein, a scale pattern is provided on the horizontal surface of the sample carrying device 01 , and the biological tissue sample M can be pressed tightly by the pressing cover 102 .

[0224] S32 , detecting the biological tissue sample M in the sample carrying device 01 to determine its position parameters in the sample carrying device 01 .

[0225] Example 1: An initial observation can be performed on the sample carrier 01, and the target area of ​​the biological tissue sample M in the sample carrier 01 can be determined based on the scale diagram set on the horizontal surface of the sample carrier 01. The specific observation method can refer to the aforementioned embodiment, and then the target area is imported into the control software of the laser imaging system 03.

[0226] Example 2: A picture of a biological tissue sample on the xy plane can be taken, and the boundary of the biological tissue sample can be identified based on the picture. The coordinate range of the biological tissue sample on the xy plane can be detected to obtain position parameters. The specific detection method refers to the previous embodiment; during imaging, the coordinate range is input into the control software of the laser imaging system, and the control software then matches its scanning range with the input coordinate range when capturing the image.

[0227] S33, using the position measuring device 04 to measure the position of the cover glass 101a of the sample carrying device 01 pressing the biological tissue sample M in the determined target area, obtain the position parameters of the biological tissue sample M in the z-axis direction, and then import the position parameters in the z-axis direction into the control software of the laser imaging system 03.

[0228] S34, place the sample carrying device 01 on the sample stage of the laser imaging system 03, control the objective lens 31 of the laser imaging system 03 to align with the target area on the xy plane during the imaging process, and perform rapid focusing in the z-axis direction according to the position parameters during the imaging process to quickly image the biological tissue sample M.

[0229] As in the technical solution of the above embodiment, the objective lens 31 of the laser imaging system 03 is controlled to quickly align with the biological tissue sample M in the xy plane during the imaging process by utilizing the observed target area, and the position parameters are used to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction, thereby achieving rapid imaging of the laser imaging system 03 and improving imaging efficiency.

[0230] An embodiment of a laser imaging system is described below.

[0231] As shown in FIG16 , FIG16 is a schematic diagram of the structure of an exemplary laser imaging system, including: an optical system 30 , a sample stage 32 , and a laser imaging device 33 ; wherein the sample stage 32 is used to place the sample carrying device 01 in any embodiment of the present application.

[0232] In one embodiment, before imaging, the sample carrier 01 can be initially observed using the positioning observation device 02, and the target region of the biological tissue sample M in the sample carrier 01 can be located based on the scale diagram of the sample carrier 01. For example, the operator can determine the position information corresponding to the biological tissue sample M using the optical microscope 21, thereby obtaining the target region where the biological tissue sample M is located.

[0233] Furthermore, a camera 22 can be set on the sample table 32 of the laser imaging system 03. The camera 22 is connected to the control software of the laser imaging system 03 through the control module 23. The control software can output instructions to control the camera 22 to take pictures of the chassis range of the sample carrier 01, wherein the control module 23 can use relevant image processing algorithms to analyze the pictures, or the control module 23 can perform certain processing on the pictures and then send the image data to the control software for processing. The control software can implant relevant algorithms to analyze the image data and determine the target area of ​​the biological tissue sample M in the sample carrier 01.

[0234] During the imaging process, the position information of the target area is imported into the control software of the laser imaging system 03. The optical system 30 controls the objective lens 31 to quickly align with the biological tissue sample M in the sample carrier 01 according to the position information, and irradiates the laser on the biological tissue sample M. The laser imaging device 33 collects the fluorescence generated on the biological tissue sample M to image the biological tissue sample M.

[0235] In one embodiment, before imaging, the position measuring device 04 measures the position of the coverslip 101a of the sample carrier 01 pressing the biological tissue sample M in a determined target area to obtain the position parameters of the biological tissue sample M in the z-axis direction, and then imports the position parameters in the z-axis direction into the control software of the laser imaging system 03.

[0236] During the imaging process, the optical system quickly focuses on the biological tissue sample M in the z-axis direction according to the position parameters and irradiates the biological tissue sample M with laser light; the laser imaging device collects the fluorescence generated on the biological tissue sample M to image the biological tissue sample M.

[0237] As described above, a sample carrier suitable for multiphoton precision imaging equipment has been designed. This carrier can be used to hold various biological tissue samples, which are then placed on a sample stage for imaging. This facilitates rapid positioning of the sample, thereby improving imaging efficiency. This also reduces the risk of contamination of the sample, shortens the time required to lock the target's z-axis focus, and enables rapid determination of the field of view focus within the xy focal plane, reducing the frequent occurrence of image blur during imaging.

[0238] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. An imaging assisting device, characterized in that: include: A sample carrying device for placing the biological tissue sample to be tested and a positioning observation device for observing the position of the biological tissue sample; The sample carrying device is used to carry biological tissue samples; The positioning observation device is used to observe the biological tissue sample on the sample carrier before imaging, and to determine the position information of the biological tissue sample on the xy plane in the sample carrier; The position information is used to control the objective lens of the laser imaging system to quickly align with the biological tissue sample during the imaging process.

2. The imaging assisting device according to claim 1, characterized in that: A ruler diagram is provided on the horizontal plane of the sample carrying device, for indicating the position information of the target object on the horizontal plane; The scale diagram includes: a coordinate ruler or grid with spatial visual reference scales; The positioning observation device determines the position information of the biological tissue sample on the xy plane in the sample carrying device according to the ruler diagram.

3. The imaging assisting device according to claim 1, characterized in that: The positioning observation device comprises an optical microscope, which is used to search for the biological tissue sample through an eyepiece and determine the position information of the biological tissue sample in the sample carrying device according to the ruler diagram.

4. The imaging assisting device according to claim 1, characterized in that: The positioning observation device includes: a camera and a control module connected thereto; The camera is used to take pictures on the horizontal plane of the sample carrying device; The control module is used to analyze the image to determine the position information of the biological tissue sample in the sample carrying device.

5. The imaging assisting device according to any one of claims 1 to 4, characterized in that: The sample carrying device comprises: a sample box, a base, a fixed cover and a ruler assembly; The sample box includes a culture dish and a pressure cover, the scale pattern is arranged on the scale assembly, and the scale assembly is connected to the bottom of the base; Wherein, the culture dish is used to carry biological tissue samples; The structure of the gland matches the culture dish and is used to cover the culture dish; The base is used to place the sample box; The fixing cover is used to fix the sample box on the base.

6. The imaging assisting device according to claim 5, characterized in that: The ruler assembly is designed as a groove, and the base is installed on the ruler assembly in an embedded manner; A positioning bead is provided on the side of the base, and a positioning hole is provided on the side of the ruler assembly; wherein the positioning bead and the positioning hole are matched and positioned.

7. An imaging assisting device, characterized in that: include: A sample carrier for placing the biological tissue sample to be tested and a position measuring device for measuring the position of the sample carrier in the z-axis direction; The sample carrying device is used to place the biological tissue sample and compress it in the z-axis direction; The position measuring device is used to measure the position of the cover glass at the bottom of the sample carrying device to obtain the position parameters of the biological tissue sample placed on the sample carrying device in the z-axis direction; The position parameter is used to control the laser imaging system to quickly focus on the biological tissue sample in the z-axis direction during the imaging process.

8. The imaging assisting device according to claim 7, characterized in that: The position measuring device is also used to measure the deformation amount of the cover glass at the bottom of the sample carrying device in the z-axis direction in real time during the process of pressing the biological tissue sample; The deformation amount is used to indicate that the biological tissue sample is compressed and placed at or near a designed focal position in the z-axis direction.

9. The imaging assisting device according to claim 7, characterized in that: Also includes: A driving device connected to the cover of the sample carrying device is used to obtain the deformation of the cover glass in real time during the process of pressing the biological tissue sample, and drive the cover of the sample carrying device to press the biological tissue sample according to the deformation, so that the biological tissue sample is placed at or near the designed focal position in the z-axis direction.

10. The imaging assisting device according to any one of claims 7 to 9, characterized in that: The position measurement device comprises: a light transmitter and a light receiver mounted on a stage, and a measurement control system connected to the light transmitter and the light receiver respectively; The light emitter is used to emit a test light beam to the cover glass at the bottom of the sample holding device; The optical receiver is used to receive the reflected light beam of the test light beam reflected by the cover glass at the bottom of the sample holding device; The measurement control system is used to calculate the focal position of the biological tissue sample in the z-axis direction according to the reflected light beam.

11. The imaging assisting device according to claim 10, characterized in that: The optical receiver is used to receive the multiple reflected light beams reflected by the test light beam from multiple surfaces of the sample carrying device; The measurement control system is also used to calculate the position coordinates of the cover glass surface of the sample supporting device in the z-axis direction in real time according to the multiple reflected light beams, and determine the deformation amount of the cover glass according to the position coordinates.

12. The imaging assisting device according to claim 11, characterized in that: The light transmitter is used to transmit a test light beam of set wavelength and brightness; The light receiver is used to simultaneously receive the reflected light beams reflected by the lower surface of the cover glass, the upper surface of the cover glass, the biological tissue sample surface and the cover pressing surface.

13. The imaging assisting device according to claim 7, characterized in that: The sample carrying device comprises: a sample box, a base and a fixed cover; The sample box includes a culture dish and a pressure cover; wherein the bottom of the culture dish is a cover glass for carrying biological tissue samples; The structure of the gland matches the culture dish and is used to cover the culture dish; The base is used to place the sample box; The fixing cover is used to fix the sample box on the base.

14. An imaging assisting device, characterized in that: include: A sample carrier for placing the biological tissue sample to be tested, a positioning observation device for observing the position of the biological tissue sample on the xy plane, and a position measurement device for measuring the sample carrier in the z-axis direction; The sample carrying device is used to place the biological tissue sample and press it in the z-axis direction; The positioning observation device is used to observe the sample carrying device before imaging to determine the position information of the biological tissue sample on the xy plane in the sample carrying device; The position measuring device is used to measure the position of the cover glass at the bottom of the sample carrying device to obtain the position parameters of the biological tissue sample placed on the sample carrying device in the z-axis direction; Wherein, the position information is used to control the objective lens of the laser imaging system to quickly align with the biological tissue sample during the imaging process; The position parameter is used to control the laser imaging system to quickly focus on the biological tissue sample in the z-axis direction during the imaging process.

15. The imaging assisting device according to claim 14, characterized in that: A ruler diagram is provided on the horizontal plane of the sample carrying device, for indicating the position information of the target object on the horizontal plane; The scale diagram includes: a coordinate ruler or grid with spatial visual reference scales; The positioning observation device determines the position information of the biological tissue sample on the xy plane in the sample carrying device according to the ruler diagram.

16. The imaging assisting device according to claim 14, characterized in that: The positioning observation device comprises an optical microscope, which is used to search for the biological tissue sample through an eyepiece and determine the position information of the biological tissue sample in the sample carrying device according to the ruler diagram.

17. The imaging assisting device according to claim 14, characterized in that: The positioning observation device includes: a camera for observing the position of the biological tissue sample on the xy plane and a control module; The camera is used to take pictures of biological tissue samples on the xy plane; The control module is used to identify the boundary of the biological tissue sample through the image, detect the coordinate range of the biological tissue sample on the xy plane, and input the coordinate range into the laser imaging system during imaging; Wherein, the laser imaging system matches its scanning range with the input coordinate range when capturing images.

18. The imaging assisting device according to claim 14, characterized in that: The position measuring device is also used to measure the deformation amount of the cover glass at the bottom of the sample carrying device in the z-axis direction in real time during the process of pressing the biological tissue sample; The deformation amount is used to indicate that the biological tissue sample is compressed and placed at or near a designed focal position in the z-axis direction.

19. The imaging assisting device according to claim 14, characterized in that: Also includes: A driving device connected to the cover of the sample carrying device is used to obtain the deformation of the cover glass in real time during the process of pressing the biological tissue sample, and drive the cover of the sample carrying device to press the biological tissue sample according to the deformation, so that the biological tissue sample is placed at or near the designed focal position in the z-axis direction.

20. The imaging assisting device according to claim 14, characterized in that: The position measurement device comprises: a light transmitter and a light receiver mounted on a stage, and a measurement control system connected to the light transmitter and the light receiver respectively; The light emitter is used to emit a test light beam to the cover glass at the bottom of the sample holding device; The optical receiver is used to receive the reflected light beam of the test light beam reflected by the cover glass at the bottom of the sample holding device; The measurement control system is used to calculate the focal position of the biological tissue sample in the z-axis direction according to the reflected light beam.

21. The imaging assisting device according to claim 20, characterized in that: The optical receiver is used to receive the multiple reflected light beams reflected by the test light beam from multiple surfaces of the sample carrying device; The measurement control system is also used to calculate the position coordinates of the cover glass surface of the sample supporting device in the z-axis direction in real time according to the multiple reflected light beams, and determine the deformation amount of the cover glass according to the position coordinates.

22. The imaging assisting device according to claim 21, characterized in that: The light transmitter is used to transmit a test light beam of set wavelength and brightness; The light receiver is used to simultaneously receive the reflected light beams reflected by the lower surface of the cover glass, the upper surface of the cover glass, the biological tissue sample surface and the cover pressing surface.

23. The imaging assisting device according to any one of claims 14 to 22, characterized in that: The sample carrying device comprises: a sample box, a base, a fixed cover and a ruler assembly; The sample box includes a culture dish and a pressure cover, the scale pattern is arranged on the scale assembly, and the scale assembly is connected to the bottom of the base; Wherein, the bottom of the culture dish is a cover glass for carrying biological tissue samples; The structure of the gland matches the culture dish and is used to cover the culture dish; The base is used to place the sample box; The fixing cover is used to fix the sample box on the base.

24. The imaging assisting device according to claim 23, characterized in that: The ruler assembly is designed as a groove, and the base is installed on the ruler assembly in an embedded manner; A positioning bead is provided on the side of the base, and a positioning hole is provided on the side of the ruler assembly; wherein the positioning bead and the positioning hole are matched and positioned.

25. A method for imaging a biological tissue sample, characterized in that: include: placing a biological tissue sample into a sample holding device; Performing an initial observation on the sample carrying device to determine a target area of ​​the biological tissue sample in the sample carrying device; The sample carrying device is placed on the sample stage of the laser imaging system, the objective lens of the laser imaging system is controlled to aim at the target area, and the biological tissue sample is quickly imaged.

26. The imaging assisting device according to claim 25, characterized in that: Performing an initial observation on the sample carrier to determine the target area of ​​the biological tissue sample in the sample carrier, including: Performing an initial observation on the sample carrying device, and determining a target area of ​​the biological tissue sample in the sample carrying device according to the scale diagram; Wherein, a scale pattern is provided on the horizontal surface of the sample carrying device.

27. The imaging assisting device according to claim 15, characterized in that: The initial observation of the sample carrying device to determine the target area of ​​the biological tissue sample in the sample carrying device includes: placing the sample holding device on the stage of the optical microscope; Searching for biological tissue samples through the eyepiece of an optical microscope; Observe the target area of ​​the biological tissue sample corresponding to the ruler map; Placing the sample carrier on the sample stage of the laser imaging system, controlling the objective lens of the laser imaging system to aim at the target area, and quickly imaging the biological tissue sample, comprises: The target area is introduced into the control software of the laser imaging system, and the control software is used to guide the objective lens of the laser imaging system to focus on the target area, so as to quickly image the biological tissue sample.

28. The imaging assisting device according to claim 25, characterized in that: The initial observation of the sample carrying device to determine the target area of ​​the biological tissue sample in the sample carrying device includes: placing a sample holding device on a sample stage of a laser imaging system; Using a camera installed on the sample stage to take a picture of the bottom plate of the sample carrier, and analyzing the picture to determine the target area of ​​the biological tissue sample in the sample carrier; Placing the sample carrier on the sample stage of the laser imaging system, controlling the objective lens of the laser imaging system to aim at the target area, and quickly imaging the biological tissue sample comprises: The target area is introduced into the control software of the laser imaging system, and the control software is used to guide the objective lens of the laser imaging system to focus on the target area, so as to quickly image the biological tissue sample.

29. A method for imaging a biological tissue sample, characterized in that: include: placing a biological tissue sample into a sample holding device; The position measuring device measures the position of the cover glass of the sample carrying device pressing the biological tissue sample, so as to obtain the position parameter of the biological tissue sample in the z-axis direction; The sample carrier is placed on the sample stage of the laser imaging system, the laser imaging system is controlled to image the biological tissue sample, and rapid focusing in the z-axis direction is performed according to the position parameters during the imaging process.

30. The method for imaging a biological tissue sample according to claim 29, characterized in that: Also includes: In the process of compressing the biological tissue sample, the biological tissue sample is compressed by a pressing cover of the sample carrying device with a set amplitude; Read the position parameters of the biological tissue sample in the z-axis direction in real time, and after waiting for a set time, until the biological tissue sample returns to a stable shape, perform the next compaction operation; The pressing operation and the reading of the position parameters of the biological tissue sample in the z-axis direction are performed cyclically until the biological tissue sample is restored to a stable shape and is placed at a focal position in the z-axis direction.

31. The method for imaging a biological tissue sample according to claim 29, characterized in that: Also includes: Performing an initial observation on the sample carrying device, and determining a target area of ​​the biological tissue sample in the sample carrying device according to a scale diagram set on a horizontal surface of the sample carrying device; During the imaging process, the objective lens of the laser imaging system is controlled to align with the target area on the xy plane to quickly image the biological tissue sample; 32. The method for imaging a biological tissue sample according to claim 29, characterized in that: Also includes: Take a picture of the biological tissue sample on the xy plane, identify the boundary of the biological tissue sample based on the picture, detect the coordinate range of the biological tissue sample on the xy plane, and input the coordinate range into the laser imaging system during imaging, so as to correspond its scanning range with the input coordinate range when taking pictures.

33. [Corrected 19.08.2024 in accordance with Rule 91] A laser imaging system, characterized in that include: An optical system, a sample stage, and a laser imaging device; wherein the sample stage is used to place the sample carrying device according to any one of claims 1 to 24; The optical system utilizes the imaging assistance device described in any one of claims 1 to 24 to assist in rapid imaging.

34. [Corrected 19.08.2024 in accordance with Rule 91] A laser imaging system according to claim 33, characterized in that During the imaging process, the optical system is used to control the objective lens to quickly align with the biological tissue sample in the sample holding device on the xy plane according to the position information, and irradiate the laser on the biological tissue sample; the laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

35. [Corrected 19.08.2024 in accordance with Rule 91] A laser imaging system according to claim 33, characterized in that During the imaging process, the optical system quickly focuses the biological tissue sample in the z-axis direction according to the position parameter and irradiates the laser on the biological tissue sample; the laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

36. [Corrected 19.08.2024 in accordance with Rule 91] A laser imaging system according to claim 33, characterized in that During the imaging process, the optical system is used to control the objective lens to quickly align the biological tissue sample in the sample carrier in the xy plane according to the position information, and to quickly focus the biological tissue sample in the z-axis direction according to the position parameter, so as to irradiate the laser on the biological tissue sample; The laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

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