Automatic liquid perfusion system and application thereof in multi-round space transcriptional group imaging

Abstract

The invention discloses an automatic liquid perfusion system and application of the automatic liquid perfusion system in multi-round space transcriptional group imaging. The automatic liquid perfusion system is formed by sequentially connecting a plurality of multi-channel switching valves connected in series, a peristaltic pump and a multi-channel switching valve. By means of the automatic liquid perfusion system, full-automatic perfusion can be achieved in the multi-round space transcriptome imaging process, perfusion of multiple samples can be achieved through sequential control, and the experiment efficiency is remarkably improved. And the sample does not need to be repeatedly detached from the imaging system in the whole process, so that subsequent data analysis is facilitated, and the imaging accuracy can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of spatial transcription group imaging, and particularly relates to a liquid automatic perfusion system for multi-round spatial transcription group imaging, and application of the liquid automatic perfusion system in multi-round spatial transcription group imaging. Background Art

[0002] Fluorescence in situ hybridization (FISH) is a molecular cell biology technique used to locate and visualize specific DNA sequences on the chromosomes of cells. Spatial transcriptomics is a technology developed in recent years that can simultaneously analyze the expression of thousands of genes on tissue sections and retain their spatial location information in the tissue. Combining FISH with spatial transcriptomics can provide detailed information about the spatial distribution of gene expression.

[0003] At present, in the process of spatial transcriptome imaging using fluorescence in situ hybridization technology, generally due to the limitation of the number of fluorescent channels of the microscopic imaging system, only one sample can be imaged at a time, and only 4 to 5 genes can be imaged at a time. In order to achieve imaging of more genes, multiple experimental operations are required, including repeated elution, staining, incubation and other operations on the same sample, which requires the sample to be repeatedly removed from the imaging system and then processed, which greatly increases the complexity and labor cost of the experiment, low experimental efficiency, and repeated disassembly and assembly of samples cannot ensure that the sample position remains unchanged during each imaging, which brings difficulties to subsequent data analysis. At the same time, the cost of the perfusion reagent required for imaging is relatively high, so it is necessary to control the amount of liquid used and minimize the dead volume of the perfusion system; and it should be noted that during the collection process, gas should not be introduced into the sample tank, otherwise it may affect the reaction of the reagent solution and the sample in the sample tank, resulting in failure of the experimental results.

[0004] Therefore, it is necessary to increase the imaging flux (i.e., the number of genes) of the spatial transcriptome, reduce the reagent cost, and improve the success rate and accuracy of the experiment. Building a fully automated spatial transcriptome imaging system can achieve this goal better and more conveniently. Therefore, a fully automated liquid perfusion system is required, and then an automated imaging acquisition system is used. By controlling them to work together, multiple rounds of spatial transcriptome imaging can be achieved. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a liquid automatic perfusion system, which can not only realize the fully automatic perfusion of reagent solutions in multiple rounds of spatial transcriptome imaging, but also improve imaging accuracy and experimental efficiency, reduce reagent costs, and facilitate subsequent data analysis.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a liquid automatic perfusion system, comprising N multi-channel switching valves and a peristaltic pump, N≥3; the multi-channel switching valve has a plurality of switching channels and a common channel;

[0008] Among them, N-1 multi-channel switching valves are connected in series in sequence to form a multi-channel series unit, and the series connection method is: the outlet of the common channel of the previous multi-channel switching valve is connected to the inlet of any switching channel of the next multi-channel switching valve, and the remaining switching channels can be connected to the liquid storage container;

[0009] The outlet of the common channel of the last multi-channel switching valve in the multi-channel series unit is connected to the inlet of the peristaltic pump, the outlet of the peristaltic pump is connected to the inlet of the common channel of the remaining multi-channel switching valve, and the outlet of the switching channel of the remaining multi-channel switching valve is connected to the sample container.

[0010] Among them, the multi-channel switching valves described in this article all have multiple switching channels (at least two) and a common channel, and when any switching channel is connected to the common channel to form a fluid channel, no fluid channel is formed between the remaining switching channels and the common channel.

[0011] In the present invention, firstly, N-1 multi-channel switching valves are connected in series in sequence to form a multi-channel series unit. Any number of multi-channel switching valves can be selected as needed, and the number of switching channels of the multi-channel switching valve can also be selected as needed, so as to achieve any number of channels and improve the perfusion efficiency and experimental efficiency. The peristaltic pump is used to provide power, and the flow rate of the liquid can be accurately controlled. At the same time, a multi-channel switching valve is set after the peristaltic pump, so as to increase multiple solution outlets, and multiple sample tanks can be connected. Multiple samples can be perfused through timing control, thereby improving the experimental efficiency.

[0012] In the present invention, the specific number of accessible sample slots can be adjusted by the number of switching channels of the multi-channel switching valve connected to the outlet of the peristaltic pump, and there is no particular limitation.

[0013] In some specific implementation cases of the present invention, a liquid automatic perfusion system is provided, which includes:

[0014] a first multi-channel switching valve;

[0015] a second multi-channel switching valve, wherein an inlet of any switching channel of the second multi-channel switching valve is connected to an outlet of the common channel of the first multi-channel switching valve;

[0016] a peristaltic pump, wherein an inlet of the peristaltic pump is connected to an outlet of the common channel of the second multi-channel switching valve;

[0017] A third multi-channel switching valve, wherein the inlet of the common channel of the third multi-channel switching valve is connected to the outlet of the peristaltic pump.

[0018] In a further embodiment, the first multi-channel switching valve and the second multi-channel switching valve each include sixteen switching channels.

[0019] In a further embodiment, the third multi-channel switching valve includes six switching channels.

[0020] It is understandable that the number of switching channels of the first, second and third multi-channel switching valves is not limited to the above cases and can be set according to specific needs.

[0021] It is understandable that the liquid automatic perfusion system also includes a control unit, which is respectively connected to each multi-channel switching valve and peristaltic pump to control each multi-channel switching valve and peristaltic pump. According to the sample needs, the corresponding program is pre-set, and the control unit is used to control the operation of each multi-channel switching valve and peristaltic pump, thereby realizing the full automatic operation of the entire perfusion operation.

[0022] The present invention further provides a multi-round spatial transcriptome imaging method, which is based on the liquid automatic perfusion system described above and comprises the following steps:

[0023] S1, emptying the gas in each pipeline of the liquid automatic perfusion system;

[0024] S2, performing a perfusion operation on the first biological sample, and performing spatial transcription group imaging and acquisition after the processing; while performing imaging and acquisition on the first biological sample, performing a perfusion operation on the second biological sample;

[0025] S3, after the imaging acquisition of the first biological sample is completed and the perfusion operation of the second biological sample is completed, the spatial transcription group imaging acquisition is performed on the second biological sample, and at the same time, a new round of perfusion operation is performed on the first biological sample (if there are only two biological samples) or a perfusion operation is performed on the third biological sample (if there are three biological samples), and so on for multiple biological samples (more than four biological samples);

[0026] S4. Repeat steps S2 to S3 until all samples and all rounds of spatial transcription group imaging are completed.

[0027] Based on the automatic liquid perfusion system described above, the perfusion and imaging operations of multiple samples are realized by setting the program to achieve timing control, thereby improving the experimental efficiency. In addition, there is no need to repeatedly disassemble and assemble biological samples during the entire experiment. The samples are always in the sample slot, which facilitates subsequent data analysis and improves imaging accuracy.

[0028] In a further embodiment, step S1 of the present invention is mainly to empty the pipes in the entire perfusion system, so as to avoid introducing air into the sample tank and affecting the sample processing. The specific operation is: switching to each reagent solution channel in turn, controlling the peristaltic pump to suck out a portion of the reagent solution, so that each reagent solution storage container and the pipe of the switching channel connected thereto are just completely filled with the reagent solution, and the gas in the pipe is emptied.

[0029] In a further solution, in order to reduce the dead volume inside the perfusion pipeline, reduce the amount of reagents used, and reduce the reagent cost, after each perfusion operation, the program controls the buffer solution inert to the perfusion reagent to push the remaining perfusion reagent in the pipeline into the sample tank.

[0030] In a further solution, the biological samples are placed in corresponding sample slots. The sample slot described in the present invention includes a closed chamber, the bottom of the chamber is a glass slide, the biological sample is attached to the glass slide, and can be directly connected to an imaging system for imaging. In addition, the chamber is connected to a liquid inlet and a liquid outlet, the liquid inlet is connected to any switching channel outlet of a multi-channel switching valve connected to the outlet of a peristaltic pump, and the liquid outlet is connected to a waste liquid tank.

[0031] The present invention further provides the application of the liquid automatic perfusion system as described above in multi-round spatial transcription group imaging. The liquid automatic perfusion system can significantly improve the experimental efficiency and accuracy of multi-round spatial transcription imaging and facilitate subsequent data analysis.

[0032] Beneficial effects of the present invention:

[0033] The liquid automatic perfusion system of the present invention can realize full-automatic perfusion in the process of multiple rounds of spatial transcriptome imaging, and the sample does not need to be repeatedly removed from the imaging system during the whole process, thereby improving the experimental efficiency and the accuracy of imaging.

[0034] In addition, the multi-channel switching valve channels in the liquid automatic perfusion system are connected in series, and any number of channels can be achieved to introduce any number of required solutions; the peristaltic pump is used to provide power, and the flow rate of the liquid can be accurately controlled. At the same time, a multi-channel switching valve is set after the peristaltic pump to increase multiple solution outlets, which can be connected to multiple sample tanks. Multiple samples can be perfused through timing control, thereby improving experimental efficiency.

[0035] The multi-round spatial transcriptome imaging based on the liquid perfusion system in the present invention can effectively evacuate the gas in the pipeline through program control, avoiding the introduction of air during the entire perfusion process to affect sample processing; at the same time, the dead volume inside the perfusion is reduced, the reagent cost is reduced, and the reagent usage is saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the connection structure of the automatic liquid perfusion system in Example 1 of the present invention.

[0037] Figure 2 Schematic diagram of the connection structure of the liquid automatic perfusion system in the multi-round spatial transcription group imaging in Example 2 of the present invention.

[0038] Figure 3 Schematic diagram of the connection structure of the liquid automatic perfusion system in the multi-round spatial transcription imaging in Example 3 of the present invention.

[0039] In the figure: 11-first multi-channel switching valve, 12-second multi-channel switching valve, 13-third multi-channel switching valve, 20-peristaltic pump, 30-liquid storage container, 41-sample tank, 411-first sample tank, 412-second sample tank, 413-third sample tank, 42-waste liquid tank. DETAILED DESCRIPTION

[0040] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] Example 1

[0043] This embodiment provides a liquid automatic perfusion system, the structure of which is as follows: Figure 1 As shown in , the liquid automatic perfusion system includes a first multi-channel switching valve 11 , a second multi-channel switching valve 12 , a peristaltic pump 20 and a third multi-channel switching valve 13 .

[0044] Among them, the first multi-channel switching valve 11 has sixteen switching channels and a common channel K1, and the second multi-channel switching valve 12 has sixteen switching channels and a common channel K2. Each inlet of the sixteen switching channels of the first multi-channel switching valve 11 is connected to a liquid storage container 30, and the outlet of its common channel K1 is connected to the inlet of any switching channel of the second multi-channel switching valve 12; each inlet of the remaining fifteen switching channels of the second multi-channel switching valve 12 is also connected to a liquid storage container 30, and the outlet of its common channel K2 is connected to the inlet of the peristaltic pump 20.

[0045] Furthermore, the third multi-channel switching valve 13 has six switching channels and one common channel K3. The outlet of the peristaltic pump 20 is connected to the inlet of the common channel K3 of the third multi-channel switching valve 13. The six switching channels of the third multi-channel switching valve 13 serve as solution outlets and are connected to the inlet of the sample tank 41. The outlet of the sample tank 41 is connected to the waste liquid tank 42.

[0046] In this embodiment, all connecting pipes are made of Teflon pipes with an inner diameter of 0.5 mm to reduce the volume of the solution in the pipes as much as possible and save the amount of reagents.

[0047] It can be understood that the automatic liquid perfusion system in this embodiment also includes a control unit (not shown in the figure), which is respectively connected to the first multi-channel switching valve 11, the second multi-channel switching valve 12, the peristaltic pump 20 and the third multi-channel switching valve 13, and is used to control the switching and action of the first multi-channel switching valve 11, the second multi-channel switching valve 12, the peristaltic pump 20 and the third multi-channel switching valve 13.

[0048] Example 2

[0049] This embodiment provides a multi-round spatial transcription group imaging method, which is based on the liquid automatic perfusion system in Example 1, and the specific steps are as follows:

[0050] 1. Pipeline connection

[0051] like Figure 2 As shown in the figure, the outlet of the common channel K1 of the first multi-channel switching valve 11 is connected to the inlet of any switching channel of the second multi-channel switching valve 12, the outlet of the common channel K2 of the second multi-channel switching valve 12 is connected to the inlet of the peristaltic pump 20, and the outlet of the peristaltic pump 20 is connected to the inlet of the common channel K3 of the third multi-channel switching valve 13.

[0052] Among them, reagent solutions A1 to A26 are reagents that need to be perfused in a single round of multi-round spatial transcription imaging, generally referring to the coding probes corresponding to the genes to be detected in spatial transcription group imaging. For example, the first round of spatial transcription group imaging requires the perfusion of reagent A1, the second round of spatial transcription group imaging requires the perfusion of reagent A2, and so on until the twenty-sixth round of spatial transcription group imaging requires the perfusion of reagent A26. Figure 2 As shown in FIG. 1 , the reagent solutions A1 to A26 are connected to 11 switching channels of the first multi-channel switching valve 11 and 15 switching channels of the second multi-channel switching valve 12 , respectively.

[0053] Furthermore, the remaining five switching channels of the first multi-channel switching valve 11 are respectively connected to reagent solutions B1~B5. In this article, reagent solutions B1~B5 will be used in each round of spatial transcription group imaging. They generally refer to sample processing reagents that are inert to reagent solution A, that is, they will not react with reagent solution A. Specific examples include flushing buffer, imaging buffer, eluent, imaging probe, etc., which can be selected according to the type of sample to be processed.

[0054] For further information, please refer to Figure 2 Any two switching channel outlets of the third multi-channel switching valve 13 are respectively connected to the inlets of the first sample slot 411 and the second sample slot 412, and the outlets of the first sample slot 411 and the second sample slot 412 are respectively connected to the waste liquid slot 42, wherein the first sample slot 411 and the second sample slot 412 are both closed liquid-perfused sample slots, which include a closed chamber for accommodating biological samples. Specifically, the bottom of the chamber is a glass slide, and the biological sample is attached to the glass slide and can be directly imaged on the imaging system, and the chamber is respectively connected to a liquid inlet and a liquid outlet.

[0055] The above connecting pipes all use Teflon pipes with an inner diameter of 0.5mm.

[0056] 2. Perfusion

[0057] (1) Through program control, switch to each reagent solution channel in turn, control the peristaltic pump to suck out a portion of the reagent solution, so that each reagent solution storage container and the pipe of the switching channel connected thereto are just completely filled with the reagent solution, and reduce the reagent coming out from the multi-channel switching valve as much as possible, which is the reagent solution that needs to be wasted;

[0058] (2) After the gas in all reagent solution storage containers and the pipes of the switching channels connected thereto is emptied, the buffer (e.g., flushing buffer) in the reagent solution B is drawn to fill the pipes connected to the common channel in the perfusion system;

[0059] (3) selecting one of the reagent solutions A1 to A26 to be perfused and the reagent solutions B1 to B5 to perform a first round of perfusion operation on the biological sample in the first sample slot 411 through program control, and after the processing is completed, performing spatial transcription group imaging and acquisition on the biological sample in the first sample slot 411; during the process of imaging and acquisition of the biological sample in the first sample slot 411, the first round of perfusion operation can be performed on the biological sample in the second sample slot 412 at the same time;

[0060] (4) After the imaging and acquisition of the biological sample in the first sample slot 411 is completed and the perfusion of the biological sample in the second sample slot 412 is completed, spatial transcription group imaging and acquisition can be performed on the biological sample in the second sample slot 412, and a new round of perfusion operation can be performed on the biological sample in the first sample slot 411;

[0061] (5) Repeat steps (3) to (4) until all samples and all rounds of spatial transcription group imaging are completed.

[0062] It should be noted that after each perfusion operation, for the reagent A with a higher cost, its usage needs to be saved as much as possible. After it is completely sucked out from the corresponding liquid storage container and the pipe of the switching channel connected thereto, in order to avoid the introduction of air, the perfusion action needs to be stopped. For the remaining reagent A in the pipe, the present invention uses a low-cost reagent B (generally a flushing buffer) that will not react with the remaining reagent A in the common pipe. The control program uses reagent B to just push reagent A into the sample tank, thereby reducing the reagent cost, saving reagent usage, and reducing the dead volume inside the perfusion system.

[0063] Example 3

[0064] This embodiment provides another multi-round spatial transcription group imaging method, which is based on the liquid automatic perfusion system in Example 1, and the specific steps are as follows:

[0065] 1. Pipeline connection

[0066] The pipeline connection in this embodiment is basically the same as that in Embodiment 2, except that a third sample tank 413 is added in the switching channel of the third multi-channel switching valve 13, and the outlet of the third sample tank 413 is connected to the waste liquid tank 42. The specific structure is as follows: Figure 3 As shown in .

[0067] 2. Perfusion

[0068] Steps (1) to (2) are the same as those in Example 2, and the gas in each pipeline in the entire system is exhausted.

[0069] (3) selecting one of the reagent solutions A1 to A26 to be perfused and the reagent solutions B1 to B5 to perform a first round of perfusion operation on the biological sample in the first sample slot 411 through program control, and after the processing is completed, performing spatial transcription group imaging and acquisition on the biological sample in the first sample slot 411; while performing imaging and acquisition on the biological sample in the first sample slot 411, performing a first round of perfusion operation on the biological sample in the second sample slot 412;

[0070] (4) After the imaging and acquisition of the biological sample in the first sample slot 411 is completed and the perfusion of the biological sample in the second sample slot 412 is completed, spatial transcription group imaging and acquisition are performed on the biological sample in the second sample slot 412, and at the same time, the first round of perfusion operation is performed on the biological sample in the third sample slot 413;

[0071] (5) After the imaging and acquisition of the biological sample in the second sample slot 412 is completed and the perfusion of the biological sample in the third sample slot 413 is completed, spatial transcription group imaging and acquisition are performed on the biological sample in the third sample slot 413, and a new round of perfusion operation is performed on the biological sample in the first sample slot 411;

[0072] (6) Repeat steps (3) to (5) until all samples and all rounds of spatial transcription group imaging are completed.

[0073] The liquid automatic perfusion system of the present invention can realize full automatic perfusion in multiple rounds of spatial transcriptome imaging, and the samples do not need to be repeatedly removed from the imaging system during the whole process, thereby improving the experimental efficiency. In addition, the number of multi-channel switching valves and the number of switching channels in the multi-channel switching valve can be designed according to needs to achieve any number of channels and introduce any number of required solutions; and any number of sample slots can be connected, and multiple biological samples can be perfused and imaged through timing control, thereby improving the efficiency of imaging experiments.

[0074] It can be understood that the simultaneous operation of 2 biological samples and 3 biological samples respectively in Examples 2 and 3 given in the present invention are only examples, and do not mean that the present invention can only realize the operation of 2 and 3 biological samples. By increasing the number of multi-channel switching valves and switching channels, any number of biological samples can be operated, which can significantly improve the experimental efficiency.

[0075] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A liquid automatic perfusion system, characterized in that: It comprises N multi-channel switching valves and a peristaltic pump, N≥3; the multi-channel switching valve has a plurality of switching channels and a common channel; Among them, N-1 multi-channel switching valves are connected in series in sequence to form a multi-channel series unit, and the series connection method is: the outlet of the common channel of the previous multi-channel switching valve is connected to the inlet of any switching channel of the next multi-channel switching valve, and the remaining switching channels can be connected to the liquid storage container; The outlet of the common channel of the last multi-channel switching valve in the multi-channel series unit is connected to the inlet of the peristaltic pump, the outlet of the peristaltic pump is connected to the inlet of the common channel of the remaining multi-channel switching valve, and the outlet of the switching channel of the remaining multi-channel switching valve is connected to the sample container.

2. The automatic liquid perfusion system according to claim 1, characterized in that: It includes: a first multi-channel switching valve; a second multi-channel switching valve, wherein an inlet of any switching channel of the second multi-channel switching valve is connected to an outlet of the common channel of the first multi-channel switching valve; a peristaltic pump, wherein an inlet of the peristaltic pump is connected to an outlet of the common channel of the second multi-channel switching valve; A third multi-channel switching valve, wherein the inlet of the common channel of the third multi-channel switching valve is connected to the outlet of the peristaltic pump.

3. The automatic liquid perfusion system according to claim 2, characterized in that: The first multi-channel switching valve and the second multi-channel switching valve each include sixteen switching channels.

4. The automatic liquid perfusion system according to claim 2, characterized in that: The third multi-channel switching valve includes six switching channels.

5. The automatic liquid perfusion system according to any one of claims 1 to 4, characterized in that: The automatic liquid perfusion system further comprises a control unit, which is respectively connected to each multi-channel switching valve and the peristaltic pump and is used to control each multi-channel switching valve and the peristaltic pump.

6. A multi-round spatial transcription group imaging method, which is based on the liquid automatic perfusion system according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, emptying the gas in each pipeline of the liquid automatic perfusion system; S2, performing a perfusion operation on the first biological sample, and performing spatial transcription group imaging and acquisition after the processing; while performing imaging and acquisition on the first biological sample, performing a perfusion operation on the second biological sample; S3, after the imaging acquisition of the first biological sample is completed and the perfusion operation of the second biological sample is completed, the spatial transcription group imaging acquisition is performed on the second biological sample, and at the same time a new round of perfusion operation is performed on the first biological sample or a perfusion operation is performed on the third biological sample, and so on for multiple biological samples; S4. Repeat steps S2 to S3 until all samples and all rounds of spatial transcription group imaging are completed.

7. The multi-round spatial transcription group imaging method according to claim 6, characterized in that: The step S1 includes the following steps: Switch to each reagent solution channel in turn, control the peristaltic pump to suck out a portion of the reagent solution, so that each reagent solution storage container and the pipe of the switching channel connected thereto are just completely filled with the reagent solution, and the gas in the pipe is emptied.

8. The multi-round spatial transcription group imaging method according to claim 6, characterized in that: The biological samples to be imaged are placed in corresponding sample slots, each of which includes a sealed chamber in which the corresponding biological sample is placed; the chamber is connected to a liquid inlet and a liquid outlet, the liquid inlet of the sample slot is connected to any switching channel outlet of a multi-channel switching valve connected to the peristaltic pump outlet, and the liquid outlet of the sample slot is connected to a waste liquid tank.

9. The multi-round spatial transcription group imaging method according to claim 6, characterized in that: After each perfusion operation is completed, the program controls the buffer solution that is inert to the perfusion reagent to push the remaining perfusion reagent in the pipeline into the sample tank.

10. Use of the liquid automatic perfusion system according to any one of claims 1 to 5 in multi-round spatial transcriptome imaging.

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