Imaging auxiliary device and laser imaging system

Abstract

The utility model relates to an imaging auxiliary device and a laser imaging system.The imaging auxiliary device comprises a sample bearing device and a position measuring device for measuring the sample bearing device in the z-axis direction, and when the sample bearing device compresses a biological tissue sample in the z-axis direction, the position measuring device measures the position of the sample bearing device in the z-axis direction; the position measuring device can be used for measuring the position of the cover glass at the bottom of the sample bearing device to obtain the position parameter of the biological tissue sample in the z-axis direction, so that the position parameter is used for controlling the laser imaging system to quickly focus the biological tissue sample in the z-axis direction in the imaging process. According to the technical scheme, rapid focusing on the biological tissue sample is facilitated, and the deformation quantity of the cover glass at the bottom of the sample bearing device in the z-axis direction can be obtained in real time, so that the biological tissue sample is indicated to be pressed, the imaging effect is greatly improved, rapid imaging of the laser imaging system is achieved, and the imaging efficiency is improved.

Description

Technical Field

[0001] This application relates to the field of optical technology, in particular to an imaging assistance device and a laser imaging system. Background Art

[0002] Multiphoton fluorescence technology is widely used for precise imaging of biological tissue samples on the premise of non-destructive samples. During the imaging process, it will not cause damage to biological tissue samples and can completely preserve various original biological information in biological tissue samples. Generally, the tissue sample is placed on a sample carrier device and then on the 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, thus completing the imaging process of the biological tissue sample.

[0003] Due to the wide variety and different sizes of biological tissue samples, the field of view (FOV) of multiphoton precision imaging equipment based on multiphoton fluorescence technology is limited. Generally, it is much smaller than the size of biological tissue samples, and it is also difficult to accurately predict the changes over time when directly placed on the sample stage. Therefore, when using multiphoton means to image tissue samples on site, imaging blurring frequently occurs, the time to lock the focal plane in the z-axis direction of the target is long, and it is difficult to quickly determine the field of view focus in the xy focal plane. Especially for some biological tissue samples with small sizes (generally within dozens to hundreds of cells), such as animal embryos, since they are much smaller than the bottom area of the sample carrier device, a microscope often needs to be used for positioning first, and then imaged on the imaging device according to the positioning data. Due to the cumbersome and time-consuming z-axis focusing of various inverted microscopes, a large range needs to be searched for focusing, so it takes a lot of time to find its position on the sample carrier device during imaging, which greatly affects the imaging operation efficiency. Summary of the Utility Model

[0004] Based on this, it is necessary to provide an imaging assistance device and a laser imaging system for at least one of the above technical defects to improve the imaging operation efficiency and imaging effect.

[0005] An imaging assistance device includes: a sample carrier device for placing a biological tissue sample to be detected and a position measurement device for measuring the position of the sample carrier device in the z-axis direction;

[0006] The sample carrier device is used to place the biological tissue sample and clamp it in the z-axis direction;

[0007] The position measurement device is used to measure the position of the cover glass at the bottom of the sample carrier device and obtain the position parameter of the biological tissue sample placed on the sample carrier device in the z-axis direction;

[0008] Among them, the position parameter is used to control the rapid focusing of the laser imaging system on the biological tissue sample in the z-axis direction during the imaging process.

[0009] In one embodiment, the position measuring device is further configured to measure the deformation of the cover glass at the bottom of the sample carrier in the z-axis direction in real time during the process of pressing the biological tissue sample;

[0010] Among them, the deformation is used to indicate that the biological tissue sample is pressed and placed at or near the focal position designed in the z-axis direction.

[0011] In one embodiment, the imaging assisting device further includes: a driving device connected to the pressing cover of the sample carrier, configured to obtain the deformation of the cover glass in real time during the process of pressing the biological tissue sample, and drive the pressing 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 focal position designed in the z-axis direction.

[0012] In one embodiment, the position measuring device includes: a light emitter and a light receiver mounted on the stage, and a measurement control system respectively connected to the light emitter and the light receiver;

[0013] The light emitter is configured to emit a test beam to the cover glass at the bottom of the sample carrier;

[0014] The light receiver is configured to receive the reflected beam reflected by the test beam passing through the cover glass at the bottom of the sample carrier;

[0015] The measurement control system is configured to calculate the focal position of the biological tissue sample in the z-axis direction according to the reflected beam.

[0016] In one embodiment, the light receiver is configured to receive multiple reflected beams reflected by the test beam passing through multiple surfaces of the sample carrier;

[0017] The measurement control system is further configured to calculate the position coordinates in the z-axis direction of the surface of the cover glass of the sample carrier in real time according to multiple reflected beams, and determine the deformation of the cover glass according to the position coordinates.

[0018] In one embodiment, the light emitter is configured to emit a test beam with a set wavelength and brightness;

[0019] The light receiver is configured to receive the reflected 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 surface of the pressing cover simultaneously.

[0020] In one embodiment, the sample carrier includes: a sample cassette, a base, and a fixed cover;

[0021] The sample cassette includes a culture dish and a pressing cover; wherein, the bottom of the culture dish is a cover glass for carrying a biological tissue sample;

[0022] The structure of the pressing cover matches that of the culture dish and is used to cover the culture dish;

[0023] The base is used to place the sample cassette;

[0024] The fixing cover is used to fix the sample cassette on the base.

[0025] In one embodiment, the imaging assistance device further includes: a positioning and observing device for observing the position of the biological tissue sample in the xy plane;

[0026] A scale 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;

[0027] The positioning and observing device is used to observe the biological tissue sample on the sample carrying device before imaging and determine the position information of the biological tissue sample in the sample carrying device according to the scale diagram;

[0028] 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.

[0029] In one embodiment, the imaging assistance device further includes: a camera for observing the position of the biological tissue sample in the xy plane and a control module;

[0030] The camera is used to take pictures of the biological tissue sample in the xy plane;

[0031] The control module is used to identify the boundary of the biological tissue sample through the picture, detect the coordinate range of the biological tissue sample in the xy plane, and input the coordinate range into the laser imaging system during imaging; wherein, the laser imaging system corresponds its scanning range to the input coordinate range when acquiring images.

[0032] A laser imaging system includes: an optical system, a sample stage, and a laser imaging device; wherein, the sample stage is used to place the sample carrying device in the imaging assistance device;

[0033] During the imaging process, the optical system quickly focuses on the biological tissue sample in the z-axis direction according to the position parameters, and irradiates the biological tissue sample with laser; the laser imaging device collects the fluorescence generated on the biological tissue sample to image the biological tissue sample.

[0034] In one embodiment, the optical system is further configured to control the objective lens to quickly align with a biological tissue sample in a sample carrier device on the xy plane according to the position information.

[0035] In the technical solutions of the above imaging assistance device and the laser imaging system, a sample carrier device and a position measurement device for measuring the sample carrier device in the z-axis direction are designed. When the sample carrier device presses a biological tissue sample in the z-axis direction, the position measurement device can measure the position of the cover glass at the bottom of the sample carrier device to obtain the position parameter of the biological tissue sample in the z-axis direction. Thus, 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. This technical solution facilitates the quick focusing on the biological tissue sample, realizes the fast imaging of the laser imaging system, and improves the imaging efficiency.

[0036] Moreover, through the position parameter measured in real time by the position measurement device, during the process of pressing the biological tissue sample, the deformation amount of the cover glass at the bottom of the sample carrier device in the z-axis direction can be obtained in real time, thereby indicating to press the biological tissue sample so that it can be more accurately placed at the focal position in the z-axis direction, greatly improving the imaging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the "optical sectioning" effect of multiphoton fluorescence;

[0038] Figure 2 is an exemplary diagram of an imaging assistance device in one embodiment;

[0039] Figure 3 is a schematic diagram of an exemplary xy coordinate scale;

[0040] Figure 4 is a schematic diagram of an exemplary grid;

[0041] Figure 5 is a schematic diagram of a positioning and observing device in one embodiment;

[0042] Figure 6 is a schematic diagram of a positioning and observing device in another embodiment;

[0043] Figure 7 is an exploded view of a sample carrier device in one embodiment;

[0044] Figure 8 is a schematic diagram of the structure of a sample carrier device in a separated state in an example;

[0045] Figure 9 is a schematic diagram of the structure of a sample carrier device in an assembled state in an example;

[0046] Figure 10Schematic diagram of an imaging assistance device according to another embodiment;

[0047] Figure 11 Schematic structural diagram of a position measurement device according to an embodiment;

[0048] Figure 12 Schematic structural diagram of a laser imaging system of an example. Detailed implementation manners

[0049] To make the objectives, technical solutions and advantages of the present application clearer and more understandable, the present application will be 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 the present application and are not used to limit the present application.

[0050] It should be noted that due to space limitations, all technical solutions cannot be enumerated. Only some implementation solutions are provided in the following embodiments. The "first" and "second" described in the present application are only used to distinguish different objects and have no substantial difference meaning.

[0051] During the imaging process of a laser imaging system using multi-photon fluorescence technology, generally, a galvanometer element is used to achieve imaging of a specific focal plane where the focus is located. As Figure 1 shown, Figure 1 is a schematic diagram of the "optical sectioning" effect of multi-photon fluorescence. As shown in the figure, the black dot position is the focus position. The imaging mode of the entire biological tissue sample is achieved by imaging through each focal plane layer by layer for accumulation, which is called the "optical sectioning" technical means for imaging. Considering the limitation of the field of view (FOV) size of the multi-photon precision imaging device, during the imaging process, the objective lens needs to accurately irradiate the laser on the biological tissue sample. Since the volume of some biological tissue samples is small, it takes a long time to search to locate the biological tissue sample. 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 on the biological tissue sample in the z-axis direction to avoid long-term imaging blurring. For this reason, the embodiments of the present application provide an imaging assistance device, which can achieve any of the following functions:

[0052] ① Provide accurate position information of the biological tissue sample in the xy plane for the laser imaging system, so that it can quickly lock the biological tissue sample in the xy plane during the imaging process; assist the laser imaging system to perform rapid imaging.

[0053] ② Provide the position parameters of the cover glass 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 to perform rapid imaging.

[0054] ③Simultaneously implement the functions of ① and ② above to assist the laser imaging system in rapid imaging.

[0055] The following describes an embodiment of the imaging assistance device for implementing the above function ①.

[0056] Refer to Figure 2 as shown, Figure 2 is an exemplary diagram of an imaging assistance device of an embodiment, including: a sample carrier device 01 and a positioning and observation device 02; wherein, the sample carrier device 01 places the biological tissue sample M to be detected, and the sample carrier device 01 is provided with a scale diagram on the horizontal plane, which can indicate the position information of the target object on the horizontal plane; the positioning and observation device 02 observes the position of the biological tissue sample M, observes the biological tissue sample M on the sample carrier device 01 before imaging, and determines the position information of the biological tissue sample M in the sample carrier device 01 according to the scale diagram; exemplarily, by using the scale diagram of the sample carrier device 01, the position information of the biological tissue sample M can be obtained through means such as eyes, magnifying glasses, microscopes, etc., so as to identify the boundary contour of the biological tissue sample M. After obtaining this position information, during the imaging process, it assists in controlling the objective lens 31 of the laser imaging system 03 to quickly align with the biological tissue sample M, thereby enabling rapid imaging and improving the imaging efficiency of the laser imaging system 03.

[0057] In one embodiment, for the scale diagram on the sample carrier device 01, it can be a coordinate scale or a grid with spatial visual reference scales, etc., as Figure 3 shown, Figure 3 is a schematic diagram of an exemplary xy coordinate scale. The position of the target object in the sample carrier device 01 can be indicated by the xy coordinate scales. As shown in the figure, the shaded area is the biological tissue sample M, and the position of the biological tissue sample M in the sample carrier device 01 can be determined through the coordinate reference scales in the figure. As Figure 4 shown, Figure 4 is a schematic diagram of an exemplary grid. The longitudinal and transverse directions of the grid are marked with scales in different sorting methods. As shown in the figure, the horizontal direction uses a, b, c..., and the vertical direction uses 0, 1, 2... etc. for marking, and the position of the biological tissue sample M in the sample carrier device 01 can be determined; for the grid, each grid cell can also be distinguished by different colors.

[0058] In one embodiment, as Figure 5 shown, Figure 5Schematic diagram of a positioning and observation device of an embodiment. The positioning and observation device 02 can adopt an optical microscope 21. The biological tissue sample M is searched out through the eyepiece 211, and the position information (such as coordinate parameters) of the biological tissue sample M in the sample carrying device 01 is determined according to the scale map. 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, in a specific case, 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) are noted down.

[0059] After obtaining the position information, it is imported into the control software of the laser imaging system 03. The scale map on the sample carrying device 01 is pre-stored in the control software of the laser imaging system 03, and the target area can be quickly aligned according to the position information, so as to assist in rapid imaging. Preferably, an optical microscope 21 with a digital output function can be adopted. The optical microscope 21 is connected to the laser imaging system 03, and the position information of the initially positioned biological tissue sample M is imported into the control software of the laser imaging system 03.

[0060] In another embodiment, as Figure 6 shown, Figure 6 Schematic diagram of a positioning and observation device of another embodiment. The positioning and observation device 02 of this embodiment includes a camera 22 and its connected control module 23. The camera 22 is arranged above the sample stage 32 of the laser imaging system 03. It should be noted that in specific implementation, the installation position of the camera 22 can be selected according to different on-site situations, and it is not limited to a specific installation position. The camera 22 is used to take pictures of the horizontal plane of the sample carrying 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 carrying device 01. Exemplarily, by taking pictures of the biological tissue sample M in the sample carrying device 01 through the camera 22, the control module 23 can automatically identify the boundary of the biological tissue sample M through the pictures 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 the scanning range on the xy plane to the input coordinate range when taking pictures.

[0061] The control module 23 of this embodiment can be connected to the laser imaging system 03. Before imaging, the sample carrier device 01 is placed on the sample stage 32. The control software of the laser imaging system 03 outputs an instruction to the control module 23 to start the camera 22 to take a picture of the sample carrier device 01. By analyzing the picture, the position information (such as coordinate parameters) of the biological tissue sample M in the sample carrier device 01 can be determined. The scale diagram of the sample carrier device 01 is pre-stored in the control software of the laser imaging system 03, and the target area can be quickly aligned according to the position information, so as to assist in rapid imaging.

[0062] Regarding the sample carrier device 01, its specific embodiments will be described below with reference to the accompanying drawings.

[0063] Figure 7 is an exploded view of the sample carrier device 01 of an embodiment. Figure 8 is a schematic structural diagram of the sample carrier device 01 in a separated state. As shown in the figure, the sample carrier device 01 of this embodiment includes: a sample cassette 10, a scale component 11, a base 12, and a fixing cover 13; wherein, the sample cassette 10 includes a culture dish 101 and a pressing cover 102. The sample cassette 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 pressing cover 102 matches that of the culture dish 101; the scale component 11 is connected to the bottom of the base 12, and the scale component 11 can be made of a transparent material; a scale diagram is marked at the hollow position of the scale component 11 relative to the base 12, and the position of the biological tissue sample M on the culture dish 101 is assisted in positioning through the scale diagram pattern.

[0064] During use, first, the biological tissue sample M to be imaged is placed into the culture dish 101 and the culture dish 101 is covered; the base 12 is used to place the sample cassette 10. After the biological tissue sample M is placed, the entire sample cassette 10 is placed on the base 12, and then the sample cassette 10 is fixed by using the fixing cover 13. Finally, the base 12 is placed on the scale component 11; after the preparation is completed, the sample carrier device 01 is placed on the sample stage 32 for imaging; before imaging, the biological tissue sample M on the sample carrier device 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 scale diagram; during imaging, the corresponding area of 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 in the xy focal plane and perform focus locking. The laser is irradiated on the biological tissue sample M through the objective lens 31, and the fluorescence generated on the biological tissue sample M is collected, and finally the biological tissue sample M is imaged.

[0065] For the sample cassette 10, it can be designed in a circular structure. There is a circular hollow part in the middle of the base 12, which can just place the sample cassette 10. Then, the sample cassette 10 is fixed by the fixing cover 13 to keep it stable. Further, the bottom of the culture dish 101 and the bottom of the pressing cover 102 are flat. The pressing cover 102 is in close contact with the culture dish 101 in an embedded manner. In order to place the biological tissue sample M on the bottom plane of the culture dish 101, a sponge pad 103 is also placed at the contact part between the pressing cover 102 and the culture dish 101. The sponge pad 103 can press the biological tissue sample M flat against the bottom plane of the culture dish 101.

[0066] In this embodiment, the sample cassette presses the biological tissue sample M flat on the bottom of the culture dish 101 through the pressing cover 102 and the sponge pad 103. Through the compaction function, the non-standard biological tissue sample M with an oversized z-axis dimension can be compacted to the z-axis height specified by the sample cassette 10, thereby shortening the time for locking the focal plane in the target z-axis direction, better correcting the scanning image hole phenomenon caused by the uneven sample, and reducing the frequent occurrence of image blurring during imaging.

[0067] For the scale component 11, preferably, it can be designed with a groove. The base 12 is installed on the scale component 11 in an embedded manner. For the convenience of disassembly and installation and fixation, positioning beads 121 are provided on the side of the base 12, and positioning holes 111 are provided on the side of the scale component 11. The positioning is performed by matching the positioning beads 121 with the positioning holes 111. During use, the base 12 is placed into the groove of the scale component 11 and fixed. When disassembling, only the base 12 needs to be pulled out.

[0068] In one embodiment, the upper part of the pressing cover 102 is provided with a handle structure 102a. Through this handle structure 102a, the pressing cover 102 can be assisted to press the biological tissue sample M onto the bottom plane of the culture dish 101. The sample cassette 10 can be a disposable component made of disposable material, which avoids the contact between the biological tissue sample M and other objects, provides good isolation from the laser imaging system 03, reduces the risk of contamination of the biological tissue sample M, and avoids the occurrence of internal and external contamination situations, meeting the relevant requirements of biosafety protection measures. The base 12 and the fixing cover 13 are reusable components, and the base 12 and the fixing cover 13 can be made of metal material. Preferably, the fixing cover 13 is installed and connected to the base 12 through threads. During use, after placing the biological tissue sample M in the sample cassette 10, the sample cassette 10 is placed into the hollow part of the base 12. A protruding part can be set on the sample cassette 10, and a card slot can be set in the base 12. The position is fixed by snapping the protruding part into the card slot, and then the fixing cover 13 is rotated and advanced by threads to firmly press the sample cassette 10 on the base 12, thereby avoiding affecting the biological tissue sample M during the movement process.

[0069] In the solution of the above embodiment, the base 12 and the fixing cover 13 can be reused. When combined with a disposable sample cassette, it can avoid the risk of contamination and ensure practicability and usage effect.

[0070] The sample carrier device 01 provided by this application is used in a detachable combination manner, with a compact structure and simple operation. As Figure 9 shown, Figure 9 FIG. is a schematic structural diagram of the sample carrier device 01 in a combined state as an example. In the usage state, the structure of the sample carrier device 01 is very compact, and the sample cassette 10 can be quickly replaced; by using the disposable sample cassette 10, good isolation and protection of the biological tissue sample M are achieved, preventing contamination, and various types of biological tissue samples M (such as under the microscope, puncture biopsy, and embryo, etc.) can be accommodated; the base 12, the fixing cover 13, and the scale assembly 11 are set to be reused. The stable base 12 and the fixing cover 13 can place the sample cassette 10 stably on the sample stage 32, avoiding any slight movement during the imaging process.

[0071] The sample carrier devices provided in the above embodiments can be used to carry various biological tissue samples. Through the scale pattern marked by the scale assembly, the position of the biological tissue sample on the culture dish can be quickly assisted in positioning, assisting the laser imaging system to quickly lock the position of the biological tissue sample, and improving the imaging operation efficiency. At the same time, the sample cassette of the sample carrier device is disposable, avoiding contact between the biological tissue sample and other objects, reducing the risk of contamination of the biological tissue sample, and meeting the relevant requirements of biosafety protection measures. The sample cassette flattens the biological tissue sample on the bottom of the culture dish through the pressing cover and the sponge pad, thereby shortening the time to lock the focus in the z-axis direction of the target and reducing the phenomenon of frequent imaging blurring during the imaging process.

[0072] For the imaging auxiliary device as in the above embodiment, when imaging a biological tissue sample, first place the biological tissue sample M into the sample carrier device 01; wherein, a scale pattern is provided on the horizontal plane of the sample carrier device 01. Among them, the bottom of the culture dish 101 can be a cover glass. For example, first place the biological tissue sample M to be imaged into the culture dish 101; place the sponge pad 103 on the biological tissue sample M; place the pressing cover 102 to cover the culture dish 101, and press the sponge pad 103 to flatten the biological tissue sample M against the bottom plane of the culture dish 101; place the entire sample cassette 10 on the base 12 and fix it with the fixing cover 13; place the base 12 on the scale assembly 11 to complete the preparation. For the scale pattern, it can be a coordinate scale or a grid with a spatial visual reference scale, etc. Of course, other forms of scale patterns can also be used to mark the position information.

[0073] The sample carrier 01 is then initially observed, and the target area of the biological tissue sample M within the sample carrier 01 is determined based on the scale diagram. The positioning observation device 02 can be used to initially observe the sample carrier 01, and the target area of the biological tissue sample M within the sample carrier 01 can be located based on the scale diagram of the sample carrier 01. For example, the sample carrier 01 can be placed on the stage of the optical microscope 21, and the biological tissue sample M can be searched for through the eyepiece 211 of the optical microscope 21. The target area of the biological tissue sample M corresponding to the scale diagram can be observed. Specifically, the operator can place the sample carrier 01 on the stage of the optical microscope 21, search for the biological tissue sample M through the eyepiece 211, and simultaneously determine the position information corresponding to the biological tissue sample M by observing the scale diagram, such as the coordinate scale, thereby obtaining the target area where the biological tissue sample M is located.

[0074] Alternatively, the sample carrier 01 may be placed on the sample stage 32 of the laser imaging system 03; a camera 22 mounted on the sample stage 32 may be used to capture an image of the base of the sample carrier 01, which may then be analyzed to determine the target region of the biological tissue sample M within the sample carrier 01. Specifically, the image may contain image content of the biological tissue sample M and a ruler image. By analyzing the image, the image region of the biological tissue sample M may be determined. This image region may then be compared with the ruler image, such as its grid position, to determine the position of the biological tissue sample M within the sample carrier 01, thereby determining the target region of the biological tissue sample M.

[0075] Finally, the sample carrier 01 is placed on the sample stage 32 of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is controlled to align with the target area, and the biological tissue sample M is quickly imaged. Specifically, based on the target area obtained by the initial positioning, 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. Exemplarily, when an optical microscope 21 is used, the target area can be imported into the control software of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is guided to focus on the target area by the control software, so as to quickly image the biological tissue sample M. In addition, if a camera 22 is used to take pictures, the target area can be correspondingly imported into the control software of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is guided to focus on the target area by the control software, so as to quickly image the biological tissue sample M.

[0076] An embodiment of an imaging assisting device for realizing the above-mentioned function ② is described below.

[0077] When the biological tissue sample M is pressed tightly by the compaction function, the time for locking the focus in the z-axis direction 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 the placement of the sample carrier device 01 each time, it is easy to cause the uncontrollable z-axis focus position of each biological tissue sample M, with a large variation range. At the same time, since the cover glass 101a at the bottom of the sample carrier device 01 is generally very thin (usually 0.17 mm), when the biological tissue sample M is loaded and the pressing cover 102 is pressed, the cover glass 101a will be bent and deformed due to different pressing degrees of the pressing cover 102, which will cause a large change in the z-axis focus of the biological tissue sample M and deviate from the focus position (the pre-designed focusing position), greatly increasing the subsequent focusing difficulty in the z-axis direction and even resulting in focusing failure in severe cases.

[0078] Therefore, in order to achieve rapid focusing on the biological tissue sample M in the z-axis direction, as Figure 10 shown, Figure 10 is an exemplary diagram of an imaging assistance device according to another embodiment. The imaging assistance device of this embodiment includes: a sample carrier device 01 for placing the biological tissue sample M to be detected and a position measuring device 04 for measuring the position of the sample carrier device 01 in the z-axis direction; wherein, the sample carrier device 01 is used to place the biological tissue sample M and press it tightly in the z-axis direction.

[0079] For the sample carrier device 01, its function is to carry the biological tissue sample M and usually includes at least a cover glass 101a and a pressing cover 102. Exemplarily, the sample carrier device 01 can include structures such as a sample cassette, a base, and a fixing cover, and specific details can refer to the relevant content of the foregoing embodiment, which will not be elaborated here.

[0080] For the position measuring device 04, it is used to measure the position of the cover glass 101a at the bottom of the sample carrier device 01 to obtain the position parameters of the biological tissue sample M placed on the sample carrier device 01 in the z-axis direction; the measured position parameters are used to control the laser imaging system 03 to rapidly focus on the biological tissue sample M in the z-axis direction during the imaging process; Exemplarily, when the position measuring device 04 performs position measurement, it can perform position measurement in the z-axis direction at multiple positions within a certain coordinate range of the biological tissue sample M in the xy plane, so as to obtain more position parameters, which are specifically determined according to the usage requirements.

[0081] As Figure 10, where the cover glass 101a at the bottom of the sample carrier 01 is prone to deformation when pressing the biological tissue sample M; exemplarily, the cover glass 101a can be the bottom of the culture dish 101 in the foregoing 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 this focal position is used during the imaging process, it will result in focusing failure. By measuring the position of the cover glass 101a of the sample carrier 01 in the z-axis direction with the position measuring device 04, an accurate position parameter, that is, the actual focal position, can be obtained.

[0082] As the technical solution of the foregoing embodiment, the measured position parameter 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.

[0083] In one embodiment, considering the differences of different biological tissue samples M, it is difficult to control the pressing degree each time when placing the biological tissue sample M in the sample carrier 01, so that the biological tissue sample M cannot accurately reach the focal position in the z-axis direction.

[0084] Therefore, for the imaging auxiliary device of this embodiment, the position parameter measured in real time by the position measuring device 04 can be used to guide the pressing of the biological tissue sample M; specifically, the position measuring device 04 is also used to measure the deformation amount of the cover glass 101a at the bottom of the sample carrier 01 in the z-axis direction in real time during the process of pressing the biological tissue sample M, and the deformation amount measured in real time is used to indicate the pressing of the biological tissue sample M so that it is placed at or near the designed focal position in the z-axis direction, as close as possible to the focal position. For example, if the pressing cover 102 is operated manually, the deformation amount of the cover glass 101a in the z-axis direction can be displayed in real time at this time, thereby guiding the accurate pressing of the biological tissue sample M.

[0085] As the technical solution of the foregoing embodiment, during the measurement, not only the final position parameter of the biological tissue sample M in the z-axis direction needs to be obtained, but also the deformation amount of the cover glass 101a can be obtained by using the position parameter measured in real time to guide the accurate pressing of the biological tissue sample M and avoid excessive pressing operation resulting in large deformation of the cover glass 101a in the z-axis direction.

[0086] In one embodiment, for the structural solution of the position measuring device 04, as shown in FIG. 11 Figure 11It is a schematic structural diagram of a position measurement device in one embodiment, which may specifically include a light emitter 41 and a light receiver 42 installed on a stage, and a measurement control system 43 connected to the light emitter 41 and the light receiver 42 respectively; 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 a reflected light beam that is 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] In one embodiment, in order to improve the control accuracy of pressing the biological tissue sample M and increase the automation efficiency, the imaging assistance device of this embodiment may further be provided with a driving device 05 to drive the pressing cover 102.

[0092] Exemplarily, the driving device 05 can be driven by a motor. The driving device 05 is connected to the pressing cover 102 of the sample carrying device 01. During the process of pressing the biological tissue sample M, by obtaining the deformation amount of the cover glass 101a in real time, the pressing cover 102 of the sample carrying device 01 is driven according to the deformation amount to press the biological tissue sample M, so that the biological tissue sample M is placed at or near the focal position designed in the z-axis direction.

[0093] As the technical solution of the above embodiment, the driving device 05 can drive the pressing cover 102 to press the biological tissue sample M more precisely, 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, facilitating subsequent imaging operation processing and improving the imaging operation efficiency.

[0094] Exemplarily, as Figure 11 , for the main electrical structure of the imaging assistance device, the measurement and control system 43 can control the light emitter 41 and the light receiver 42 to perform ranging, and is connected to the driving device 05 to control the driving device 05 to press the pressing cover 102 through the position data measured in real time.

[0095] As the technical solution of the above embodiment, by emitting a test beam with specific parameters, receiving the reflected beams reflected by multiple target surfaces, and analyzing the distances of different target surfaces, the position parameters of the biological tissue sample M and the deformation amounts of each component of the sample carrying device 01 can be accurately obtained.

[0096] The embodiments of the imaging assistance device for implementing the above function ③ are described below.

[0097] In one embodiment, the imaging assistance 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 and observing device for observing the position of the biological tissue sample M in the xy plane.

[0098] For the sample carrying device 01, it can be used to place the biological tissue sample M and press it in the z-axis direction. A scale diagram is provided on the horizontal plane of the sample carrying device 01 to indicate the position information of the target object on the horizontal plane.

[0099] For the positioning and observation device, it can be used to observe the biological tissue sample M on the sample carrier device 01 before imaging, and determine the position information of the biological tissue sample M on the xy plane in the sample carrier device 01 according to the scale map; 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.

[0100] For the position measuring device 04, it can be used to measure the position of the cover glass 101a at the bottom of the sample carrier device 01, and obtain the position parameter of the biological tissue sample M placed on the sample carrier device 01 in the z-axis direction; the position parameter is 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.

[0101] Exemplarily, for the specific structures and functions of the sample carrier device 01, the positioning and observation device, and the position measuring device 04, reference can be made to the previous embodiments, which will not be elaborated here.

[0102] For the imaging assistance device as described in the above embodiments, 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 on the xy plane during the imaging process, and the position parameter is used to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction, so as to realize the rapid imaging of the laser imaging system 03 and improve the imaging efficiency.

[0103] For the imaging assistance device as described in the above embodiments, when imaging the biological tissue sample, the biological tissue sample M can be first placed in the sample carrier device 01; the biological tissue sample M can be pressed tightly by the pressing cover 102. Then, the position measuring device 04 is used to measure the position of the cover glass 101a of the sample carrier device 01 pressing the biological tissue sample M, and obtain the position parameter of the biological tissue sample M in the z-axis direction. Exemplarily, when the sample carrier device 01 is placed on the stage of the optical microscope 21 to observe and search for the biological tissue sample M, the position parameter in the z-axis direction is obtained according to the observed target area of the biological tissue sample M and by using the position measuring device 04 at the same time. Finally, the sample carrier device 01 is placed on the sample stage 32 of the laser imaging system 03, and the laser imaging system 03 is controlled to image the biological tissue sample M, and rapid focusing in the z-axis direction is performed according to the position parameter during the imaging process. Exemplarily, the position parameter can be imported into the control software of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is guided by the control software to quickly focus on the biological tissue sample M in the z-axis direction.

[0104] According to the technical solution of the above embodiment, the position measuring device 04 measures the position of the cover glass 101a to obtain the position parameter of the biological tissue sample M in the z-axis direction. Thus, when the laser imaging system 03 performs imaging, it can perform rapid focusing in the z-axis direction, improving the imaging efficiency.

[0105] In order to more precisely press the biological tissue sample M in the sample carrier device 01 so that it is close to the cover glass 101a and placed at or near the focal position designed in the z-axis direction. In one embodiment, when pressing the biological tissue sample M, the pressing cover 102 of the sample carrier device 01 can press the biological tissue sample M with a set amplitude. Read the position parameter of the biological tissue sample M in the z-axis direction in real time to guide the pressing operation. Stop the pressing operation and wait for a set time (such as about 5 s) until the biological tissue sample M returns to a stable shape. Determine whether the biological tissue sample M is placed (close to) the focal position in the z-axis direction. If so, perform the next pressing operation; otherwise, read the last position parameter of the biological tissue sample M in the z-axis direction and import it into the control software of the laser imaging system 03 for imaging.

[0106] According to the technical solution of the above embodiment, by reading the position parameter of the biological tissue sample M in the z-axis direction in real time to guide the pressing operation, it is possible to avoid excessive pressing operation resulting in too large a deformation of the cover glass 101a, which affects the rapid focusing operation of the biological tissue sample M and improves the imaging efficiency.

[0107] In addition, for the imaging auxiliary device of the above embodiment, when imaging the biological tissue sample, it can first place the biological tissue sample M into the sample carrier device 01; wherein, a scale diagram is provided on the horizontal plane of the sample carrier device 01, and the biological tissue sample M can be pressed by the pressing cover 102. Then, detect the biological tissue sample M in the sample carrier device 01 to determine its position parameter in the sample carrier device 01. Next, the position measuring device 04 measures the position of the cover glass 101a pressing the biological tissue sample M in the sample carrier device 01 in the determined target area to obtain the position parameter of the biological tissue sample M in the z-axis direction, and then import the position parameter in the z-axis direction into the control software of the laser imaging system 03. Finally, place the sample carrier 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 in the xy plane during imaging, and perform rapid focusing in the z-axis direction according to the position parameter during imaging to rapidly image the biological tissue sample M.

[0108] For detecting and determining its position parameter in the sample carrier device 01, the following two examples can be adopted:

[0109] Example 1: An initial observation can be made on the sample carrier device 01, and the target area of the biological tissue sample M in the sample carrier device 01 can be determined according to the scale diagram set on the horizontal plane of the sample carrier device 01. For the specific observation method, reference can be made to the foregoing embodiments. Then, the target area is imported into the control software of the laser imaging system 03.

[0110] Example 2: A picture of the biological tissue sample on the xy plane can be taken. According to the picture, the boundary of the biological tissue sample is identified, and the coordinate range of the biological tissue sample on the xy plane is detected to obtain the position parameters. For the specific detection method, reference can be made to the previous embodiments; during imaging, the coordinate range is input into the control software of the laser imaging system, so that the control software can correspond its scanning range to the input coordinate range when acquiring images.

[0111] As the technical solution of the above embodiments, by using the observed target area to control the objective lens 31 of the laser imaging system 03 to quickly align with the biological tissue sample M on the xy plane during the imaging process and using the position parameters to control the laser imaging system 03 to quickly focus on the biological tissue sample M in the z-axis direction, the rapid imaging of the laser imaging system 03 can be realized, and the imaging efficiency can be improved.

[0112] The embodiments of the laser imaging system are described below.

[0113] As Figure 12 shown, Figure 12 is a schematic structural diagram of an example 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 carrier device 01 in any embodiment of the present application.

[0114] In one embodiment, before imaging, the positioning and observation device 02 can be used to make an initial observation on the sample carrier device 01, and the target area of the biological tissue sample M in the sample carrier device 01 can be located according to the scale diagram of the sample carrier device 01. For example, the operator can determine the position information corresponding to the biological tissue sample M through the optical microscope 21, so as to obtain the target area where the biological tissue sample M is located.

[0115] Furthermore, a camera 22 can also be provided on the sample stage 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 device 01. Among them, the control module 23 can use relevant image processing algorithms to analyze the pictures, or after the control module 23 performs certain processing on the pictures, the image data is sent to the control software for processing. The control software can implant relevant algorithms to analyze the image data to determine the target area of the biological tissue sample M in the sample carrier device 01.

[0116] During the imaging process, the position information of the target area is imported into the control software of the laser imaging system 03, and the optical system 30 controls the objective lens 31 to quickly align with the biological tissue sample M in the sample carrier device 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.

[0117] In one embodiment, before imaging, the position measuring device 04 measures the position of the cover glass 101a that presses the biological tissue sample M in the determined target area to obtain the position parameter of the biological tissue sample M in the z-axis direction, and then imports the position parameter in the z-axis direction into the control software of the laser imaging system 03.

[0118] During the imaging process, the optical system quickly focuses on the biological tissue sample M in the z-axis direction according to the position parameter, and irradiates the laser on the biological tissue sample M; the laser imaging device collects the fluorescence generated on the biological tissue sample M to image the biological tissue sample M.

[0119] As described in the above technical solution, a sample carrier device suitable for multi-photon precision imaging equipment is designed, which can be used to carry various biological tissue samples and then placed on the sample stage for imaging detection, facilitating the rapid locking of the position of the biological tissue sample, thereby improving the imaging operation efficiency. At the same time, it also reduces the risk of contamination of the biological tissue sample, shortens the time to lock the focus in the z-axis direction of the target, and realizes the rapid determination of the field of view focus in the xy focal plane, reducing the frequent occurrence of imaging blurring during the imaging process.

[0120] The above embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the utility model patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. An imaging assistance device, characterized in that, Including: A sample carrier for placing a biological tissue sample to be detected and a position measuring device for measuring the sample carrier in the z-axis direction; The sample carrier 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 carrier, and obtain the position parameter of the biological tissue sample placed on the sample carrier in the z-axis direction; Wherein, 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.

2. The imaging assistance device according to claim 1, wherein The position measuring device is also used to measure the deformation amount of the cover glass at the bottom of the sample carrier in the z-axis direction in real time during the process of compressing the biological tissue sample; Wherein, the deformation amount is used to indicate that the biological tissue sample is compressed and placed at or near the focal position designed in the z-axis direction.

3. The imaging assistance device according to claim 1, characterized in that, Also including: A driving device connected to the pressing cover of the sample carrier, which is used to obtain the deformation amount of the cover glass in real time during the process of compressing the biological tissue sample, and drive the pressing cover of the sample carrier to compress the biological tissue sample according to the deformation amount, so that the biological tissue sample is placed at or near the focal position designed in the z-axis direction.

4. The imaging assistance device according to any one of claims 1 to 3, characterized in that The position measuring device includes: a light emitter and a light receiver installed on the stage, and a measurement control system respectively connected to the light emitter and the light receiver; The light emitter is used to emit a test beam to the cover glass at the bottom of the sample carrier; The light receiver is used to receive the reflected beam reflected by the test beam passing through the cover glass at the bottom of the sample carrier; 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 beam.

5. The imaging assistance device according to claim 4, wherein The light receiver is used to receive multiple reflected beams reflected by the test beam passing through multiple surfaces of the sample carrier; The measurement control system is also used to calculate the position coordinates of the cover glass surface of the sample carrier in the z-axis direction in real time according to multiple reflected beams, and determine the deformation amount of the cover glass according to the position coordinates.

6. The imaging assistance device according to claim 5, characterized in that, The light emitter is used to emit a test beam with a set wavelength and brightness; The light receiver is used to simultaneously receive the reflected 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 surface of the pressing cover.

7. The imaging assistance device according to claim 1, characterized in that, Also including: a positioning observation device for observing the position of the biological tissue sample in the xy plane; A scale diagram is provided on the horizontal plane of the sample carrier, which is used to indicate the position information of the target object on the horizontal plane; The positioning observation device is used to observe the biological tissue sample on the sample carrier before imaging, and determine the position information of the biological tissue sample in the sample carrier according to the scale diagram; 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.

8. The imaging assistance device according to claim 1, characterized in that, Also including: a camera for observing the position of the biological tissue sample in the xy plane and a control module; The camera is used to take pictures of the biological tissue sample in the xy plane; The control module is used to identify the boundary of the biological tissue sample through 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; wherein, the laser imaging system corresponds its scanning range to the input coordinate range during image acquisition.

9. A laser imaging system, characterized in that, It includes: an optical system, a sample stage, and a laser imaging device; wherein, the sample stage is used to place the sample carrier device in any one of the imaging auxiliary devices according to claims 1-8; During imaging, the optical system quickly focuses on 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.

10. The laser imaging system according to claim 9, wherein A scale diagram is provided on the horizontal plane of the sample carrier device for indicating the position information of the target object on the horizontal plane; the optical system is also used to quickly align the objective lens with the biological tissue sample in the sample carrier device on the xy plane according to the position information.

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