Automatic collecting and sorting device for slices in three-dimensional imaging process of sample

Abstract

The invention relates to an automatic slice collecting and sorting device in a sample three-dimensional imaging process, which comprises a slice collecting unit and a slice sorting unit, the slice collecting unit is used for automatically collecting slices generated in the sample three-dimensional imaging process, and the slice sorting unit is used for sorting the collected slices. The slice collecting unit and the slice sorting unit are arranged at the same time, the slices can be automatically collected, the target slices and the non-target slices can be automatically sorted, and the sorting efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an automatic collection and sorting device for slices during three-dimensional imaging of samples. Background Art

[0002] In recent years, the developed automated microscopic optical imaging technology combines optical tomography technology with precision cutting technology to achieve sub-micron resolution three-dimensional fine imaging of large samples of centimeter-sized biological tissues such as mouse brains. The specific steps include: First, methods such as virus labeling / transgenic are used to label target structures such as neurons and blood vessels in biological tissues; then the biological tissue is embedded in an embedding medium such as resin to form a sample to achieve a hardness that enables precision cutting with a micron thickness; finally, the sample is fixed in a water tank filled with a processing fluid, and after obtaining a tomographic image of the shallow part of the entire sample section through scanning by an objective lens and an imaging module, the imaged part on the surface of the sample is cut off using a tool. By continuously repeating the process of "sample section imaging - sample surface cutting", the three-dimensional fine structure information of a large sample with a centimeter size can be obtained.

[0003] During this imaging process, collecting the generated sample slices and using them for subsequent omics sequencing and other analyses can further provide richer sample information and has great research value. However, during the three-dimensional imaging of large-size biological tissue samples that lasts for several hours or even several days, the number of generated slices is in the tens of thousands. Selecting slices containing target cells, structures, or regions from them requires manual on-site monitoring and repeated manual slice collection operations, which are inefficient, time-consuming, and laborious. Therefore, it is necessary to design a device that can automatically collect slices during the three-dimensional imaging of samples.

[0004] Existing slice collection devices mainly use a conveyor belt roller type with adhesion or electrostatic adsorption. A conveyor belt is used to attach the sample slices generated by a slicing machine, and a conveyor belt that collects and stores the attached slices through structures such as rollers is used. Although this method can achieve automatic slice collection, it is not suitable for collecting slices generated in an aqueous environment. Therefore, a fluid collection scheme has been proposed, using water flow to collect slices through a waterway into a storage structure. However, the waterway structure and working mode of existing schemes are complex, and efficient sorting of a large number of slices cannot be achieved, that is, discarding invalid slices (non-target slices) and only retaining slices (target slices) containing target cells, structures, or regions. Summary of the Invention

[0005] The first objective of the present invention is to be able to achieve both automatic slice collection and slice sorting. To achieve this objective, the present invention provides the following technical solutions:

[0006] An automatic collection and sorting device for slices during the three-dimensional imaging of a sample, comprising a slice collection unit and a slice sorting unit. The slice collection unit is used to automatically collect the slices generated during the three-dimensional imaging of the sample, and the slice sorting unit is used to sort the collected slices.

[0007] In the above solution, by simultaneously setting up the slice collection unit and the slice sorting unit, not only can the automatic collection of slices be achieved, but also the automatic sorting of target slices and non-target slices can be realized, improving the sorting efficiency.

[0008] In an implementable solution, the slice collection unit includes a tank body, a cutter, a delivery pipe, and a water pump. The tank body is used to hold the processing liquid in which the sample is immersed. The cutter is used to cut the sample to form slices. One end of the delivery pipe is connected to the water pump, and the other end faces the cutter to suck the slices formed by the cutter cutting the sample.

[0009] In the above solution, the tank body holds the processing liquid, the sample is immersed in the processing liquid. After the cutter slices the sample, the water pump sucks the slices with the processing liquid into the delivery pipe, thereby realizing the automatic collection of slices. The structure is simple and the cost is low.

[0010] The second object of the present invention is to reduce the complexity of the pipeline structure. To achieve this object, the present invention provides the following technical solutions:

[0011] The slice sorting unit includes a sorting pipe, a filter, and a collector. One end of the sorting pipe is connected to the water pump, and is used to sort the target slices collected in the delivery pipe into the collector, and sort the non-target slices collected in the delivery pipe into the filter.

[0012] In the above solution, the sorting pipe is connected to the water pump, and the slices in the delivery pipe can enter the sorting pipe. Then the target slices directly enter the collector, and the non-target slices directly enter the filter. It can not only realize the automatic sorting of slices, but also the pipeline structure of the whole device is simple, and the control method is simple. It only needs to control the slices in the delivery pipe to enter the filter or the collector. Moreover, the flow mode of the slices with the processing liquid in the pipeline is a unidirectional downstream flow, and it is not easy to block the pipeline.

[0013] In a more optimized solution, the slice sorting unit further includes a filter branch pipe, a sorting branch pipe, a first solenoid valve, and a second solenoid valve. One end of the filter branch pipe is connected to the filter, and the other end is connected to the sorting pipe. The first solenoid valve is installed on the filter branch pipe; one end of the sorting branch pipe is connected to the sorting pipe, and the other end faces the collector. The second solenoid valve is installed on the sorting branch pipe.

[0014] In the above solution, the solenoid valve cooperates with the branch pipe, which can more accurately achieve slice sorting, prevent non-target slices from entering the collector, and also prevent target slices from entering the filter, ensuring the accuracy and reliability of sorting.

[0015] In a further optimized solution, photodetectors are installed on the conveying pipe, the filtering branch pipe, and the sorting branch pipe for detecting and counting slices.

[0016] In the above solution, by arranging photodetectors in the pipeline, when a slice passes by, it can be detected and counted. This can not only achieve slice quantity statistics, but also control the working duration of the water pump according to the feedback signal of the photodetector to prevent the pipeline from being blocked by slices.

[0017] In an implementable solution, the collector includes a collecting plate, and a number of holes are provided on the collecting plate, and a filter screen is arranged at each hole.

[0018] In a further optimized solution, there are multiple sorting branch pipes, and the distance between two adjacent sorting branch pipes is equal to the distance between two adjacent holes in the collecting plate.

[0019] In the above solution, by setting multiple sorting branch pipes, multiple slices can be sorted at one time, which can reduce the frequency of moving the collecting plate, thereby improving the sorting efficiency. Moreover, the distance between two adjacent sorting branch pipes is equal to the distance between two adjacent holes in the collecting plate, which can ensure that the slices accurately enter the holes in the collecting plate and guarantee the collection success rate.

[0020] In a further optimized solution, the collector further includes a liquid collecting tank for collecting the processing liquid that passes through the filter screen.

[0021] In the above solution, by setting the liquid collecting tank, the processing liquid that passes through the filter screen can enter the liquid collecting tank and then be discharged through the liquid collecting tank. This can not only ensure the effectiveness of slice collection, but also avoid affecting the slice collection efficiency due to excessive accumulated processing liquid.

[0022] In a further optimized solution, it further includes a return pipe. The filter and the liquid collecting tank are connected to the return pipe, and the return pipe is connected to the tank body.

[0023] In the above solution, the processing liquid collected by the filter and the liquid collecting tank is recycled to the tank body through the return pipe, which can realize the recycling of the processing liquid, avoid the additional collection of this part of the processing liquid, and reduce costs.

[0024] In a further optimized solution, it further includes an X-Y translation stage for driving the liquid collecting tank and the collecting plate to move so as to align different holes in the collecting plate with the sorting branch pipes.

[0025] In the above solution, by arranging the X-Y translation stage and driving the liquid collecting tank and the collecting plate to move through the X-Y translation stage, manual movement is avoided, full-automatic sorting and collection of slices are realized, the working efficiency is improved, and the accuracy of the moving position can be guaranteed by precisely controlling the moving amount.

[0026] Compared with the prior art, the present invention has the following technical advantages:

[0027] (1) Based on the slice collection and sorting device of the present invention, it is possible to judge whether the current slice contains target cells according to the real-time imaging result, so as to discard invalid slices. During the long-term three-dimensional imaging process of samples, accurate and automatic sorting and collection of a small number of sample slices containing target cell bodies from a large number of slices can be realized without manual attendance, greatly improving the working efficiency.

[0028] (2) Based on the slice collection and sorting device of the present invention, the waterway structure is simple, and a gentle and unidirectional flowing liquid is used as the transportation carrier, which can realize the efficient sorting and collection of sample slices with a thickness of micron level, soft and easy to break, and ensure the integrity of the slices.

[0029] (3) Based on the slice collection and sorting device of the present invention, the overall structure is simple, without complex sensors, having good long-term operation stability, low use cost, and being easy to maintain regularly.

[0030] For other advantages of the present invention, please refer to the relevant descriptions in the embodiment part. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a schematic structural diagram of an automatic collection and sorting device for slices during the three-dimensional imaging process of samples exemplified in the embodiments of the present invention.

[0033] Figure 2 For application Figure 1 The working flow chart of collecting and sorting slices by the shown device.

[0034] Figure 3 It is a schematic diagram of cell body recognition in the sample cross-sectional image based on the embodiments of the present invention.

[0035] Figure 4 It is an image of the sample cross-sectional imaging result and the corresponding collected slice based on the embodiments of the present invention.

[0036] Figure 5 The sliced images are obtained after collecting the sample slices contained in 20 consecutive coronal planes of a mouse brain sample.

[0037] Attachment Figure 1 In the [attachment], the component names represented by each label are as follows:

[0038] 1. Imaging module;

[0039] 21. Tank; 211. Processing fluid; 212. Sample; 22. Tool; 23. Delivery pipe; 24. Water pump;

[0040] 31. Sorting pipe; 311. Filter branch pipe; 312. Sorting branch pipe; 32. Filter; 321. Flow direction of non-target slices; 33. Collection plate; 331. Flow direction of target slices; 34. First solenoid valve; 35. Second solenoid valve; 36. Liquid collection tank; 37. X-Y translation stage; 38. Return pipe; 381. Flow direction of the liquid in the return pipe;

[0041] 4. Computer; 41. Photoelectric detector; 42. Imaging data; 43. Control signal; 44. Feedback signal. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention 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 invention and are not used to limit the present invention.

[0043] Please refer to Figure 1 , an automatic collection and sorting device for slices during the three-dimensional imaging of a sample provided in this embodiment includes a slice collection unit and a slice sorting unit. The slice collection unit is used to automatically collect the slices generated during the three-dimensional imaging of the sample, and the slice sorting unit is used to sort the collected slices into target slices and non-target slices. A target slice refers to a slice containing target cells, structures or regions and can be used for subsequent analysis, and a non-target slice refers to a useless slice, that is, a slice other than the target slice.

[0044] In order to facilitate the identification of target slices and non-target slices, the automatic collection and sorting device provided in this embodiment needs to be used in cooperation with an imaging module and a computer. The imaging module 1 is used to collect images of the sample cross-section, and the computer 4 is used to receive the images collected by the imaging module 1 and identify target slices and non-target slices.

[0045] Can refer to Figure 1, in this embodiment, the slice collection unit includes a tank body 21, a cutter 22, a delivery pipe 23, and a water pump 24, which are used to cut samples and collect slices. There is machining fluid 211 in the tank body 21, and the machining fluid 211 is water. The sample 212 is a resin-embedded mouse brain sample. The sample 212 is fixed in the tank body 21 and is completely immersed in the machining fluid 211. The cutter 22 uses a diamond cutter and is installed above the sample 212 to cut the sample 212 to form slices. The width of the cutter 22 is smaller than the cross-sectional width of the sample 212. Therefore, only one slice strip can be cut off in one cutting. A complete cross-section of the sample 212 contains multiple slice strips. The delivery pipe 23 is fixed above the cutting edge of the cutter 22. One end is immersed in the machining fluid 211 and faces the cutter. This end is beveled to facilitate sucking the slices more conveniently. The other end is connected to the water pump 24. The water pump 24 uses a peristaltic pump and is used to suck the machining fluid containing slices.

[0046] In this embodiment, the water pump is a peristaltic pump. This kind of pump squeezes the rubber hose to deform through a rotor to push the water flow in the hose forward. For slices with a small size (such as slices of resin-embedded samples with a width of several millimeters), they can pass through the peristaltic pump smoothly under the drive of the water flow without breaking or being damaged.

[0047] Please continue to refer to Figure 1 , in this embodiment, the slice sorting unit includes a sorting pipe 31, a filter 32, a collector, a first solenoid valve 34, and a second solenoid valve 35. One end of the sorting pipe 31 is connected to the water pump 24, and the other end is connected to a tee, branching out a filtering branch pipe 311 and a sorting branch pipe 312. The filtering branch pipe 311 is connected to the filter 32. The first solenoid valve 34 is installed on the filtering branch pipe 311 and is used to control the non-target slices to enter the filter 32; the sorting branch pipe 312 is located above the collector. The second solenoid valve 35 is installed on the sorting branch pipe 312 and is used to control the target slices to enter the collector. Both the first solenoid valve 34 and the second solenoid valve 35 use electromagnetic pinch valves, and the pipes at the solenoid valves are all made of silicone hoses. The first solenoid valve 34 and the second solenoid valve 35 can be automatically opened and closed under the control of a computer. If no solenoid valve is set, manual control is required, which is not conducive to precise control.

[0048] The collector includes a collection plate 33 and a liquid collection tank 36. The collection plate 33 is a perforated plate, that is, a number of holes are provided on the collection plate, and a filter screen is arranged at each hole. The liquid collection tank 36 is arranged below the collection plate 33. The machining fluid with slices flows into the holes of the collection plate 33, the slices remain on the filter screen, and the machining fluid flows through the filter screen into the lower liquid collection tank 36. Through the liquid collection tank 36, the machining fluid can be effectively collected, ensuring the cleanliness and hygiene of the environment, and is more conducive to the collection of slices on the filter screen.

[0049] The filter 32 and the liquid collecting tank 36 are connected with a liquid return pipe 38, and the liquid return pipe 38 is connected to the tank body 21. The installation heights of the filter 32 and the liquid collecting tank 36 are both higher than that of the tank body 21. The processing liquid in the filter 32 and the liquid collecting tank 36 can flow back into the tank body 21 by itself under the action of gravity, avoiding the need to arrange additional power structures, simplifying the structure and reducing costs at the same time.

[0050] One hole in the collecting plate 33 can only collect one slice. Therefore, during the slice collection process, the collecting plate 33 needs to be continuously moved to align the idle hole with the nozzle of the sorting branch pipe. In order to achieve fully automated collection and sorting, the collecting plate 33 can be fixed on the X-Y translation stage 37 (the X-Y translation stage 37 refers to a mechanism that can move along the X-axis and the Y-axis. The two double-headed arrows in the figure respectively represent the movement directions along the X-axis and the Y-axis). The X-Y translation stage 37 drives the collecting plate 33 to move, so as to align different holes of the collecting plate 33 with the sorting branch pipes 312 for separately packing different slices into different holes in sequence. In this embodiment, in order to facilitate the installation of the X-Y translation stage 37, the collecting plate 33 is fixed on the liquid collecting tank 36, and the liquid collecting tank 36 is fixed on the X-Y translation stage 37. The X-Y translation stage 37 drives the liquid collecting tank 36 to move, and the collecting plate 33 can follow the liquid collecting tank 36 to move synchronously.

[0051] The conveying pipe 23, the filtering branch pipes 311 and the sorting branch pipes 312 are all made of transparent polytetrafluoroethylene hard pipes, with smooth pipe walls and low friction coefficients, and have excellent corrosion resistance. Photoelectric detectors 41 are installed on the pipes for detecting and counting the slices, and sending the detection signals to the computer 4. The computer 4 can control the working mode of the water pump according to the detection signals of the photoelectric detectors, which helps to eliminate the situation of slice blockage in the pipeline.

[0052] Multiple sorting branch pipes 312 can be connected to the sorting pipe 31, so as to reduce the moving distance of the X-Y translation stage 37 and improve the sorting efficiency.

[0053] Figure 2 The following is the flowchart of the work for slice collection and sorting in this embodiment. The detailed description of the working process of the device in this embodiment will be given below in combination with the various components of the device in this embodiment:

[0054] Step S1: Start the imaging module and the computer to start data acquisition;

[0055] Step S2: The imaging module 1 scans and images the sample cross-section, and transmits the imaging data 42 to the computer 4;

[0056] Step S3: The computer 4 generates a sample cross-section image and performs cell body recognition;

[0057] Step S4: The computer 4 determines whether the slice to be cut contains the target cells, that is, determines whether the slice is the target slice;

[0058] Step S5: If the target cells are included, i.e., the section is a target section, the computer 4 sends control signals 43 to the first solenoid valve 34 and the second solenoid valve 35, closes the first solenoid valve 34, and opens the second solenoid valve 35. The section will flow along the flow direction 331 of the target section to the designated hole on the collection plate 33; if the target cells are not included, i.e., the section is a non-target section, the first solenoid valve 34 is opened and the second solenoid valve 35 is closed, and the section will flow along the non-target section flow direction 321 to the filter 32;

[0059] Based on Figure 1 the structure shown, the sections in the water flow can be collected and sorted along one water flow direction driven by the peristaltic pump. Not only Figure 1 the structure of the device shown is simple, but also the collection success rate is high.

[0060] Step S6: The sample is moved to the cutter 22, the water pump 24 is started, and cutting begins. The computer 4 monitors the feedback signal 44 output by the photodetector 41;

[0061] Step S7: The computer 4 determines whether the section has been collected to the designated position or filtered according to the feedback signal 44 of the photodetector 41. If the photodetector 41 detects that the section has been collected to the designated position or filtered, the next step is executed; if the photodetector 41 does not detect the section, the water pump 24 continues to rotate for a certain time to clean the residual sections in the pipeline, and then the next step is executed; on the basis that the sections themselves flow unidirectionally, combined with such a control method, it is possible to effectively prevent the sections from blocking the pipeline and ensure the smooth collection and sorting of the sections.

[0062] Step S8: The water pump 24 is turned off, the computer 4 records the collection situation, and the X-Y translation stage 37 is moved to prepare for the next collection;

[0063] Step S9: The computer 4 determines whether the imaging of the entire sample is completed according to the cross-sectional image; if the imaging of the entire sample is not completed, the sample is moved to the starting point of the next cross-sectional imaging, and returns to Step S2; if the imaging of the entire sample is completed, Step S10 is executed;

[0064] Step S10: Stop data acquisition and section collection.

[0065] Figure 3 It is a result diagram for cell body recognition of the real-time imaging result of the sample cross-section. The sample used is a mouse brain sample with sparse labeling of primary somatosensory cortex neurons by AAV-GFP virus. Figure 3 (A) is the generated cross-sectional imaging result. By using Gaussian filtering to remove the interference of autofluorescence signals and noise signals in the image, and then using the dynamic threshold segmentation method and morphological operations, accurate cell body extraction and positioning can be achieved, asFigure 3 as shown in (B), and the processing process meets the real-time requirement.

[0066] Figure 4 are the imaging results of the sample cross-section and the images of the corresponding collected slices. After the cross-section of the mouse brain sample is scanned and imaged, a diamond tool is used to slice the sample cross-section to obtain 2 sample slices with a width of 2.4 mm and a thickness of 8 μm. Figure 4 (A) is the imaging result of the sample cross-section obtained by the imaging module 1 (real-time staining with propidium iodide nucleic acid dye); Figure 4 (B) and (C) are the fluorescence imaging results and bright-field imaging results of the propidium iodide nucleic acid staining of the collected sample slices. It can be seen that the mouse brain coronal plane corresponds to two strip-shaped slice samples, both of which are intact and can achieve a good correspondence with the cross-section imaging results.

[0067] Figure 5 is the slice imaging display result after collecting the sample slices contained in 20 consecutive coronal planes of the mouse brain sample. Each mouse brain coronal plane contains 2 sample slices with a width of 2.4 mm and a thickness of 8 μm. A total of 40 sample slices are automatically collected, all of which are intact, proving that the present invention has a very high collection success rate and can maintain the integrity of the slices.

[0068] The above-described embodiments are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications, substitutions, and improvements, etc. These modifications, substitutions, and improvements should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. An automatic collection and sorting device for slices in the process of three-dimensional imaging of samples, characterized in that: It comprises a slice collecting unit and a slice sorting unit. The slice collecting unit is used to automatically collect slices generated during the three-dimensional imaging process of the sample, and the slice sorting unit is used to sort the collected slices.

2. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 1, characterized in that: The slice collection unit includes a trough body, a tool, a delivery pipe, and a water pump. The trough body is used to hold processing fluid without immersion of samples. The tool is used to cut samples to form slices. One end of the delivery pipe is connected to the water pump, and the other end faces the tool to absorb the slices formed by the tool cutting the sample.

3. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 2, characterized in that: The slice sorting unit includes a sorting tube, a filter and a collector. One end of the sorting tube is connected to the water pump and is used to sort the target slices collected in the conveying tube into the collector, and to sort the non-target slices collected in the conveying tube into the filter.

4. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 3, characterized in that: The slice sorting unit also includes a filtering branch pipe, a sorting branch pipe, a first solenoid valve, and a second solenoid valve. One end of the filtering branch pipe is connected to the filter, and the other end is connected to the sorting pipe. The first solenoid valve is installed on the filtering branch pipe; one end of the sorting branch pipe is connected to the sorting pipe, and the other end faces the collector. The second solenoid valve is installed on the sorting branch pipe.

5. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 4, characterized in that: The conveying pipe, the filtering branch pipe and the sorting branch pipe are all equipped with photoelectric detectors for detecting and counting slices.

6. The automatic collection and sorting device for slices in the process of three-dimensional imaging of a sample according to claim 4, characterized in that: The collector comprises a collecting plate, a plurality of holes are arranged on the collecting plate, and a filter screen is arranged at each hole.

7. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 6, characterized in that: There are multiple sorting branch pipes, and the distance between two adjacent sorting branch pipes is equal to the distance between two adjacent holes in the collecting plate.

8. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 6, characterized in that: The collector also includes a liquid collecting tank for collecting the processing fluid that passes through the filter screen.

9. The automatic collection and sorting device for slices in the process of three-dimensional imaging of samples according to claim 8, characterized in that: It also includes a liquid return pipe, the filter and the liquid collecting tank are connected to the liquid return pipe, and the liquid return pipe is connected to the tank body.

10. The automatic collection and sorting device for slices in the process of three-dimensional imaging of a sample according to claim 8, characterized in that: It also includes an XY translation stage for driving the liquid collecting tank and the collecting plate to move so as to align different holes in the collecting plate with the sorting branch pipe.

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