A high-throughput cell imaging method and application based on fluorescence barcoding immunolabeling

Abstract

The present invention discloses a high-throughput cell imaging method and application based on fluorescent barcoded immunolabeling. A fluorescence barcoded immunolabeling signal amplification method based on the HCR reaction is used to construct ADC complexes corresponding to various proteins in cells. The antibody end recognizes the target, and the DNA end triggers the HCR reporting signal. Multiple proteins in cells are simultaneously incubated and labeled for fluorescence imaging. The artificial + computer image intelligent recognition combination is used for multi-color differentiation of cells to distinguish and quantify multi-protein fluorescence imaging, which is used for multi-protein imaging detection and analysis in vitro tumor cells. The present invention realizes simultaneous imaging of multiple proteins in cells based on fluorescence reporting signals under different combinations, solves problems such as cumbersome operation and cell denaturation caused by repeated imaging, and at the same time combines artificial and image intelligent recognition to achieve the differentiation and quantitative analysis of each protein, which has important application value in the fields of multi-target and multi-omics analysis.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a high-throughput cell imaging method and application based on fluorescent barcoded immunolabeling. Background Art

[0002] The imaging-based protein localization method is one of the current techniques for localizing and quantitatively analyzing proteins at the subcellular scale, and plays an important role in exploring the complex structure and function of the proteome. The programmable DNA immunolabeling technology has been successfully applied to single-cell protein imaging analysis. Through antibody DNA labeling of the target protein and subsequent fluorescence signal output, multiple rounds of imaging analysis of multiple proteins in a single cell can be performed through repeated "imaging-displacement" cycles. However, due to the cumbersome operation and problems such as cell deformation and signal distortion caused by repeated imaging, there is still room for improvement.

[0003] The hybridization chain reaction (HCR) was first proposed by R.M. Dirks and N.A. Pierce et al. in 2004, and is an isothermal signal amplification technology based on DNA strand displacement reaction. In the HCR system, the target molecule triggers the alternating opening of two DNA stem-loops, and self-assembles to obtain a linear double-stranded DNA nanostructure containing a large number of repeating units, which has the advantages of constant temperature, enzyme-free, and high amplification efficiency, and is widely used in the fields of biosensing, bioimaging, and biomedicine. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a method for constructing a fluorescence barcoded immunolabeling signal amplification method (iSAFE-HCR) based on the HCR reaction, using click chemistry principles to synthesize antibodies that initiate DNA cross-linking of the primer strand, forming an antibody-primer complex (ADC), and further using the ADC to trigger the HCR reaction, and its output signal as a reporter tag.

[0005] Another purpose of the present invention is to provide the application of the above iSAFE-HCR method in the simultaneous imaging of multiple protein targets in cells.

[0006] A third purpose of the present invention is to provide a high-throughput cell imaging method based on fluorescent barcoded immunolabeling. The above iSAFE-HCR method is used to construct ADC complexes corresponding to each protein in the cell. By recognizing the target through the antibody end and triggering the HCR reporter signal through the DNA end, multiple proteins in the cell are simultaneously incubated and labeled and fluorescence imaging is performed to outline the overall cell morphology.

[0007] The fourth object of the present invention is to provide the application of the above-mentioned high-throughput cell imaging method based on fluorescent barcoded immunolabeling in the detection and analysis of multi-protein imaging in vitro tumor cells. By using the above-mentioned high-throughput cell imaging method based on fluorescent barcoded immunolabeling, the multi-color differentiation technology of cells combined with artificial and computer image intelligent recognition is used to distinguish and quantify the fluorescence results of multi-protein imaging, and a multi-color composite image of each corresponding protein is drawn for analysis. For each target, its corresponding signal tag is determined, and the purpose of accurate recognition and analysis is achieved according to the imaging signal.

[0008] To achieve one of the above objects, the technical solution adopted by the present invention is:

[0009] The present invention provides a method for constructing a fluorescence barcoded immunolabeling signal amplification method based on the HCR reaction, including: using the click chemical reaction principle to cross-link the DBCO-modified initiator strand DNA (T strand) with the azide-modified antibody to synthesize an antibody primer complex (ADC). The T strand specifically triggers the HCR reaction, and the output fluorescence signal is used as a reporter tag to label the target; that is, using the click chemistry principle to synthesize the antibody cross-linked with the initiator strand DNA. One antibody corresponds to one initiator strand DNA, and one initiator strand DNA can trigger a group of HCR reactions for signal amplification and output results. In theory, n targets can be labeled with n antibodies and correspond to n signal output results.

[0010] Preferably, taking a group of HCR reaction elements as an example, the HCR reaction elements are four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures, and their sequences are shown in the following table. The 3'-end modified fluorescent groups are Pacific Blue, FAM, TAMRA, and Cy5, and the excitation wavelengths are 405nm, 488nm, 560nm, and 633nm respectively, and are pseudocolor-labeled with blue, green, yellow, and red respectively;

[0011]

[0012] Different situations generated by different combinations of color, combination, or ratio are used as the reporter tags of each target to distinguish the signal differences output by different targets.

[0013] To achieve the second of the above objects, the technical solution adopted by the present invention is:

[0014] The present invention provides the application of the above-mentioned iSAFE-HCR method in the simultaneous imaging of multi-protein targets in cells.

[0015] To achieve the third of the above objects, the technical solution adopted by the present invention is:

[0016] The present invention provides a high-throughput cell imaging method based on fluorescent barcoded immunolabeling. The iSAFE-HCR method is used to construct ADC complexes corresponding to various proteins in cells. The target is recognized by the antibody end, and the HCR reporter signal is triggered by the DNA end. Multiple proteins in cells are simultaneously incubated and labeled for fluorescence imaging to outline the overall cell morphology.

[0017] Preferably, taking the simultaneous incubation and imaging of 6 proteins as an example, the sequences of 6 groups of initiating strands and fluorescent reporter elements are shown in the following table:

[0018] Table 2: Sequences of 6 groups of initiating strands and fluorescent reporter elements

[0019]

[0020]

[0021] It includes the following steps:

[0022] Step a: Design specific sequences of signal reporter tags for 6 protein targets in HeLa cells, and modify fluorescent groups on different hairpins. The sequences are shown in Table 2.

[0023] Step b: React the DBCO-modified T strands, that is, 6 groups of T strands, with 6 groups of target antibodies modified with azide in PBS buffer. Due to the principle of click chemistry, the antibody cross-links with DNA to obtain ADC complexes for initiating 6 groups of HCR reactions.

[0024] Step c: Incubate the cross-linked antibodies obtained in step b simultaneously in fixed HeLa cells, and perform HCR reactions simultaneously in fixed HeLa cells using the 6 groups of H1, H2, H3, and H4 strands obtained in step a.

[0025] Step d: The incubated HeLa cells obtained in step c are subjected to confocal microscopy to obtain multi-color imaging maps of multiple proteins in the same HeLa cell.

[0026] Preferably, the specific steps of step b are as follows: In PBS buffer with a total volume of 100 μL, the antibody corresponding to the target and the NHS-PEG4-N3 cross-linker are magnetically stirred at 1000 rpm for 2 h at room temperature according to a molar ratio of 30 μM:1.5 mM, and the azide-modified antibody is obtained by ultrafiltration and centrifugal purification; the azide-functionalized antibody is incubated with the DBCO-modified T strand at a molar ratio of 10 μM:100 μM at 4 °C for 1 day while magnetically stirring at 1000 rpm. The excess T strand is removed by ultrafiltration and centrifugation, and the antibody cross-linked with the T strand is resuspended with 1×PBS to obtain the T strand-labeled antibody; the 6 groups of T strands correspond to 6 antibodies in sequence, and 6 DNA-modified ADC complexes for immunolabeling are prepared in the same manner.

[0027] Preferably, the specific steps of step c are as follows: Culture 1×10 4 HeLa cells in a confocal dish and place them in a cell incubator at 37°C with 5% CO2 for 24 hours; discard the culture medium the next day, wash three times with pre-cooled PBS, fix at room temperature with 4% paraformaldehyde for 15 minutes, and wash five times with PBS; add 500 μL of 0.5% TritonX-100 to the dish, incubate at 37°C for 20 minutes, and wash 3 times with PBS; then add 500 μL of 5% BSA to the dish, incubate at room temperature for 30 minutes, and wash 3 times with PBS; incubate the 6 groups of ADCs obtained in step b simultaneously overnight at 4°C, and wash 3 times with PBS; then add 6 groups of H1, H2, H3, and H4 chains with a final concentration of 500 nM simultaneously, react at 37°C for 1.5 hours, and after the reaction, wash 3 times with PBS.

[0028] To achieve the fourth above-mentioned object, the technical solution adopted by the present invention is:

[0029] The present invention provides the application of the above-mentioned high-throughput cell imaging method based on fluorescent bar-coded immunolabeling in the detection and analysis of multi-protein imaging in vitro tumor cells. The above-mentioned high-throughput cell imaging method based on fluorescent bar-coded immunolabeling is used to perform fluorescence imaging on tumor cells. Combining the "manual + computer-aided" two-step method, first, the cell positions corresponding to each protein target are roughly distinguished manually, and then a computer image intelligent recognition software is used to perform computer-aided color extraction of each protein in multiple excitation channels.

[0030] Preferably, the tumor cells include but are not limited to HeLa cells, A549 cells, and HepG2 cells.

[0031] Preferably, taking HeLa cells as an example of tumor cells, the above-mentioned high-throughput cell imaging method based on fluorescent bar-coded immunolabeling is used to perform fluorescence imaging on tumor cells, and simultaneous imaging analysis of 6 protein targets is completed under four excitation light paths. The specific steps are as follows:

[0032] Step 1: Manually select and frame according to the determined positions of each protein in the cell to obtain a rough multi-color map of each protein;

[0033] Step 2: Use a computer image intelligent recognition software to select a region of interest (ROI) in each protein, perform color extraction for the four excitation channels on the original image (image_roi), and copy an image (image_fill) for subsequent conversion to RGB format for coloring;

[0034] Step 3: For the image_roi image, set it as different grayscale images in each channel, calculate the corresponding grayscale values, which are used as the average fluorescence values of each protein in this channel, add them to the ROI Manager for quantitative analysis;

[0035] Step 4: According to the four pseudo-color code, perform hexadecimal-RGB conversion, namely red (#ec141f; 236, 20, 31), green (#1fcc16; 31, 204, 22), blue (#0e6cfa; 14, 108, 250), yellow (#fad60e; 250, 214, 14), and color the regions in the ROI Manager in image_fill;

[0036] Step 5: Through Process->Image Calculator, superimpose the original image and the labeled image.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: Immunolabel cells with antibodies that initiate chain DNA cross-linking, and use the four-hairpin-mediated HCR signal output as a reporter tag to construct an iSAFE-HCR method to achieve one-to-one detection and analysis of target-antibody-output signals. While amplifying the target signal, it can also increase the number of simultaneous imaging of cellular proteins, achieving the purpose of high-throughput analysis. After excluding the influence of fluorescence crosstalk, signal outputs under different combinations and ratios of four fluorescences can be obtained, achieving the purpose of simultaneous imaging of multiple proteins in cells, solving problems such as cumbersome operations and cell denaturation caused by repeated imaging. At the same time, combining artificial and image intelligent recognition to achieve the differentiation and quantitative analysis of each protein. Compared with the multi-round repeated "imaging-replacement" cyclic imaging, multiple targets can be imaged and analyzed simultaneously in the same time and space, outlining the full state of cells, which is helpful to achieve multi-dimensional, efficient, and accurate imaging, and has important application value in the fields of multi-target and multi-omics analysis. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of cell protein imaging using the iSAFE-HCR method in the embodiment; by constructing an ADC complex, it can specifically recognize the target on the one hand and trigger the HCR self-assembly reaction on the other hand to achieve the purpose of signal amplification.

[0039] Figure 2 It is a pseudo-color diagram under the combination of four different excitation light-emitting fluorescent groups in the embodiment (9 combinations are selected from single coding to four coding respectively).

[0040] Figure 3 It is a diagram of the actual protein imaging results of triggering 9 different coding cases with actin as an example in the embodiment.

[0041] Figure 4 It is a diagram of the orthogonality analysis results of triggering the corresponding HCR signals for groups T1-T6 with actin as an example in the embodiment.

[0042] Figure 5The figure shows the results of simultaneous imaging of 6 proteins in HeLa cells in the example.

[0043] Figure 6 The figure is a flow analysis diagram of the cell multi-color discrimination technology in the example.

[0044] Figure 7 The figure is an actual analysis diagram of simultaneous imaging of 6 proteins in HeLa cells using the cell multi-color discrimination technology in the example. Detailed implementation manners

[0045] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0046] Example 1

[0047] Confocal characterization image of the four-fluorescence combination DNA multi-coded HCR signal in intracellular actin protein

[0048] React the DBCO-modified T strand (Trigger DNA-DBCO) with the azide-modified actin antibody in PBS buffer. Due to the click chemistry principle, the antibody can crosslink with the DNA, thus obtaining the actin-T complex ( Figure 1 ) for initiating the HCR reaction. The specific operation is as follows:

[0049] Prepare a 200 μL ep tube. In 100 μL of PBS buffer in total volume, mix the actin antibody and the NHS-PEG4-N3 crosslinker at a molar ratio of 30 μM:1.5 mM. Subsequently, stir magnetically at room temperature for 2 h (1000 rpm), and purify through a 10 kd ultrafiltration centrifugal column to obtain the azide-modified actin antibody. Then incubate with the DBCO-modified T strand at a molar ratio of 10 μM:100 μM at 4 °C for 1 day while stirring magnetically (1000 rpm). The excess T strand is removed by ultrafiltration centrifugation, and the antibody crosslinked with the T strand is resuspended with 1×PBS to obtain the T strand-labeled antibody (actin-T complex). Prepare 9 confocal dishes, culture 1×10 4 HeLa cells in them. Discard the culture medium the next day, and after fixation, punching, and blocking steps, add the above actin-T complex, incubate overnight at 4 °C, and recover the antibody the next day. After washing 3 times with PBS, add 4 μL of H1, H2, H3, and H4 respectively. Different groups of H1, H2, H3, and H4 are modified with different fluorescent molecules:

[0050] The first group: H1, H2, H3, and H4 are respectively modified with FAM, 0, 0, 0;

[0051] Group 2: H1, H2, H3, and H4 modify Cy5, 0, 0, 0 respectively;

[0052] Group 3: H1, H2, H3, and H4 modify TAMRA, 0, 0, 0 respectively;

[0053] Group 4: H1, H2, H3, and H4 modify Pacific Blue, 0, 0, 0 respectively;

[0054] Group 5: H1, H2, H3, and H4 modify FAM, Cy5, 0, 0 respectively;

[0055] Group 6: H1, H2, H3, and H4 modify TAMRA, Pacific Blue, 0, 0 respectively;

[0056] Group 7: H1, H2, H3, and H4 modify FAM, TAMRA, 0, 0 respectively;

[0057] Group 8: H1, H2, H3, and H4 modify Cy5, TARMA, Pacific Blue, 0 respectively;

[0058] Group 9: H1, H2, H3, and H4 modify FAM, Cy5, TARMA, Pacific Blue( Figure 2 )

[0059] Make up to 200 μL with 1×PBS, mix well, place in a small dish, and react at 37°C for 1.5 h. After the reaction, wash 3 times and take confocal images under a 100× oil immersion objective. The 4 different fluorescence excitation channels are FAM-488, Cy5-633, TAMRA-560, and Pacific Blue-405. The same fluorescence shooting settings are used for all images. The results are as Figure 3 shown.

[0060] From Figure 3It can be seen that by using actin-T for multicolor encoding imaging, different color combinations from single encoding to quadruple encoding can be achieved. Through simple channel analysis, it can be obtained that in the first group, there is only the green false color of FAM; in the second group, there is only the red false color of Cy5; in the third group, there is only the yellow false color of TAMRA; in the fourth group, there is only the blue false color of Pacific Blue; in the fifth group, there are the green and red false colors of FAM and Cy5; in the sixth group, there are the yellow and blue false colors of TAMRA and Pacific Blue; in the seventh group, there are the green and yellow false colors of FAM and TAMRA; in the eighth group, there are the red, yellow, and blue false colors of Cy5, TARMA, and Pacific Blue; in the ninth group, there are the green, red, yellow, and blue false colors of FAM, Cy5, TARMA, and Pacific Blue. This shows that the above several fluorescence barcoding combinations can be effectively imaged under a single target, and the composite color differences under each combination are large and easy to distinguish, which is beneficial to the simultaneous analysis of subsequent multi-protein imaging.

[0061] Example 2

[0062] Application of the iSAFE-HCR method in the simultaneous imaging analysis of 6 proteins in HeLa cells in the present invention

[0063] React the DBCO-modified T strand (Trigger DNA-DBCO) with the azide-modified antibody in PBS buffer. Due to the principle of click chemistry, the antibody can crosslink with DNA, thus obtaining the ADC complex for initiating the HCR reaction. The specific operation is as follows:

[0064] Prepare six 200 μL ep tubes, labeled 1, 2, 3, 4, 5, and 6 respectively. Mix 6 kinds of protein antibodies (actin, Ezrin, LaminA, GM130, FBLR, Histone H3) with the NHS-PEG4-N3 crosslinker at a molar ratio of 30 μM:1.5 mM. Subsequently, stir magnetically at room temperature for 2 h (1000 rpm), and purify through an ultrafiltration centrifugal column to obtain the azide-modified antibody respectively.

[0065] Subsequently, incubate with the DBCO-modified T1-T6 strands at a molar ratio of 10 μM:100 μM at 4 °C for 1 day while stirring magnetically (1000 rpm). Remove the excess T strands by ultrafiltration centrifugation, and resuspend the antibody crosslinked with the T strand with 1×PBS to obtain the T strand-labeled antibody (ADC complex). In this process, the actin-T1 to actin-T6 complexes are synthesized respectively for subsequent orthogonality verification, as well as Ezrin-T2, LaminA-T3, GM130-T4, FBLR-T5, and Histone H3-T6. Prepare multiple confocal dishes, and place 1×10 4HeLa cell culture. Among them, the culture medium was discarded on the second day. After fixation, perforation, and blocking steps, in the orthogonality verification, actin-T1 to actin-T6 complexes were added respectively, and incubated overnight at 4°C. The antibodies were recovered the next day. After washing 3 times with PBS, 4 μL of each of the corresponding H1, H2, H3, and H4 chains in 6 groups were added respectively (final concentration was 500 nM, only one kind of fluorescence modification). In multi-color imaging, 6 groups of complexes including actin-T1, Ezrin-T2, LaminA-T3, GM130-T4, FBLR-T5, and Histone H3-T6 were added simultaneously, and incubated overnight at 4°C. The antibodies were recovered the next day. After washing 3 times with PBS, 4 μL of each of the corresponding H1, H2, H3, and H4 chains in 6 groups were added simultaneously (final concentration was 500 nM). React at 37°C for 1.5 h, and after the reaction, wash 3 times with PBS. Place under a 100× oil immersion objective to take confocal images. Subsequently, combining the "manual + computer-aided" two-step method, first make a rough distinction (manually) for the cell positions corresponding to each protein target, and then use the computer image intelligent recognition software image J to extract the colors of the four excitation channels for each protein (computer-aided). For each target, determine its corresponding signal tag, and achieve accurate recognition and analysis according to the imaging signal. The results are as Figure 4 , Figure 5 , Figure 7 .

[0066] It can be seen from Figure 4 that the HCR reactions between groups Q1-Q6 are specific. In the presence of the corresponding T chain, the reaction is triggered to show fluorescence, while no fluorescence can be observed in non-corresponding groups, indicating that it is helpful for the imaging reaction of multiple proteins in subsequent simultaneous imaging applications. Subsequently, the 6 groups of proteins were imaged simultaneously. As shown in Figure 5 , the full state of the cell with multiple color identifications was successfully outlined. From the outer cell membrane to the innermost nucleolus, there are corresponding colors for characterization and distinction, and the differences are obvious, which is conducive to analysis. Finally, according to the cell multi-color differentiation technology ( Figure 6 ), the corresponding 6 proteins can be extracted from the multi-color composite image, and the specific fluorescence conditions of the ROI therein can be further analyzed, that is, the cell membrane Ezrin only has fluorescence in the TAMRA channel; the cytoskeleton actin only has fluorescence in the Cy5 channel; LaminA only has fluorescence in the Pacific Blue channel; the Golgi GM130 has fluorescence in both FAM and TAMRA channels; the nucleolus FBLR has fluorescence in both FAM and Cy5 channels; the cell nucleus Histone H3 has fluorescence in Cy5, TAMRA, and PacificBlue channels, and the fluorescence ratios are consistent, which conforms to the theoretical assumption ( Figure 7) It shows that the high-throughput imaging method of cells based on fluorescence barcoding immunolabeling in the present invention can successfully achieve the simultaneous incubation imaging analysis of multiple proteins in cells, which is simple and clear, and avoids the cumbersome steps of the past repeated "imaging - washing" cycles.

Claims

1. A fluorescence barcoding immunoassay signal amplification method based on HCR reaction, characterized in that Its construction method includes: crosslinking DBCO-modified primer strand DNA with azide-modified antibody to synthesize an antibody primer complex. The primer strand DNA serves as the T strand, and the T strand specifically triggers the HCR reaction. The output fluorescence signal serves as a reporter tag to label the target, obtaining a fluorescence barcoding immunolabeling signal amplification method based on the HCR reaction. The HCR reaction elements are four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures, and their sequences are shown in SEQ ID NO:1-4 respectively. The 3'-ends are modified with fluorescent groups Pacific Blue, FAM, TAMRA, and Cy5 respectively, and the excitation wavelengths are 405 nm, 488 nm, 560 nm, and 633 nm respectively, and are pseudo-colored with blue, green, yellow, and red respectively. The T strand sequence is shown in SEQ ID NO:

5. Different situations generated by different combinations of colors or ratios serve as reporter tags for each target to differentially distinguish the signals output for different targets.

2. A high-throughput cell imaging method based on fluorescence barcoding immunolabeling, characterized in that, Using the fluorescence barcoding immunolabeling signal amplification method based on the HCR reaction described in claim 1 to construct antibody primer complexes corresponding to each protein in the cell. The target is recognized by the antibody end and the HCR reporter signal is triggered by the DNA end. Multiple proteins in the cell are simultaneously incubated and labeled for fluorescence imaging to outline the overall cell morphology. The cell is a HeLa cell, and 6 proteins are simultaneously incubated and imaged, including the following steps: Step a: Design specific sequences of 6 groups of signal reporter tags for 6 protein targets in HeLa cells, and modify fluorescent groups on different hairpins. The sequences of the 6 groups of primer strands and fluorescent reporter elements are as follows: Group Q1: The T strand sequence is shown in SEQ ID NO:5, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:7-10 respectively; Group Q2: The T strand sequence is shown in SEQ ID NO:11, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:12-15 respectively; Group Q3: The T strand sequence is shown in SEQ ID NO:16, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:17-20 respectively; Group Q4: The T strand sequence is shown in SEQ ID NO:21, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:22-25 respectively; Group Q5: The T strand sequence is shown in SEQ ID NO:26, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:27-30 respectively; Group Q6: The T strand sequence is shown in SEQ ID NO:31, and the sequences of the four hairpin DNA strands H1, H2, H3, and H4 with stem-loop structures are shown in SEQ ID NO:32-35 respectively; Step b: React 6 sets of T strands with 6 sets of azide-modified target antibodies in PBS buffer. The antibody crosslinks with the DNA to obtain an antibody primer complex for initiating 6 sets of HCR reactions; Step c: Incubate the crosslinked antibodies obtained in step b simultaneously in fixed HeLa cells, and perform HCR reactions on the fixed HeLa cells simultaneously with the 6 sets of H1, H2, H3, and H4 strands obtained in step a; Step d: The incubated HeLa cells obtained in step c are subjected to confocal imaging to obtain a multi-color imaging map of multiple proteins in the same HeLa cell.

3. The high-throughput cell imaging method based on fluorescence barcoding immunolabeling according to claim 2, wherein The steps of step b are as follows: In PBS buffer with a total volume of 100 μL, react the antibody corresponding to the target with the NHS-PEG4-N3 crosslinker at a molar ratio of 30 μM : 1.5 mM and stir magnetically at 1000 rpm at room temperature for 2 h. Ultrafiltration centrifugation purification is used to obtain azide-modified antibodies; The azide-functionalized antibodies are incubated with DBCO-modified T strands at a molar ratio of 10 μM : 100 μM at 4 °C for 1 day while stirring magnetically at 1000 rpm. The excess T strands are removed by ultrafiltration centrifugation, and the T strand-crosslinked antibodies are resuspended with 1×PBS to obtain T strand-labeled antibodies; The 6 sets of T strands correspond to 6 antibodies in sequence, and 6 DNA-modified antibody primer complexes for immunolabeling are prepared in the same manner.

4. The high-throughput cell imaging method based on fluorescence barcoding immunolabeling according to claim 2, wherein The steps of step c are as follows: Add 1×10 4 HeLa cells were cultured in confocal dishes and placed in a cell incubator at 37 °C and 5% CO2 for 24 h; the culture medium was discarded the next day, washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and washed five times with PBS; 500 μL of 0.5% TritonX-100 was added to the dish, incubated at 37 °C for 20 min, and washed 3 times with PBS; then 500 μL of 5% BSA was added to the dish, incubated at room temperature for 30 min, and washed 3 times with PBS; the 6 groups of antibody primer complexes obtained in step b were incubated simultaneously overnight at 4 °C and washed 3 times with PBS; then 6 groups of H1, H2, H3, and H4 chains with a final concentration of 500 nM were added simultaneously, reacted at 37 °C for 1.5 h, and after the reaction, washed 3 times with PBS.

5. Application of the high-throughput cell imaging method based on fluorescent barcoding immunolabeling according to any one of claims 2-4 in the detection and analysis of multi-protein imaging in vitro tumor cells. The high-throughput cell imaging method based on fluorescent barcoding immunolabeling is used to perform fluorescence imaging on tumor cells, combined with an artificial + computer-aided two-step method. First, the cell positions corresponding to each protein target are roughly distinguished manually, and then a computer image intelligent recognition software is used to perform computer-aided color extraction of each protein in multiple excitation channels.

6. The application according to claim 5, characterized in that The tumor cells are HeLa cells. The high-throughput cell imaging method based on fluorescent barcoding immunolabeling is used to perform fluorescence imaging on HeLa cells, and simultaneous imaging analysis of 6 protein targets is completed under four excitation light paths. The steps are as follows: Step 1: Manually select and frame according to the determined positions of each protein in the cell to obtain a rough multi-color map of each protein; Step 2: Use a computer image intelligent recognition software to select an ROI area in each protein, perform color extraction for the image_roi image for the four excitation channels, and copy an image_fill image for subsequent conversion to the RGB format for coloring; Step 3: For the image_roi image, set it as different grayscale images in each channel, calculate the corresponding grayscale values, which serve as the average fluorescence values of each protein in this channel, and add them to the ROI Manager for quantitative analysis; Step 4: Perform hexadecimal-RGB conversion according to the four pseudocolor codes, and color the area in the ROIManager in the image_fill image; Step 5: Overlay the original image with the annotated image through Process -> Image Calculator.

Images