CN113960001A

CN113960001A - AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof

Info

Publication number: CN113960001A
Application number: CN202111211348.1A
Authority: CN
Inventors: 陈同生; 高璐
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2022-01-21
Anticipated expiration: 2041-10-18
Also published as: CN113960001B

Abstract

The invention discloses an AutoQT-FRET method based on a correction factor of a primary imaging measurement system and application thereof. The method comprises the steps of firstly utilizing a single-transfer plasmid cell sample to obtain the three-channel average fluorescence intensity I of each plasmid through E-FRET imaging_DD、I_AA、I_DAAnd acceptor-sensitized fluorescence intensity F_CAnd with I_DDAnd F_CDrawing a scatter diagram for the horizontal and vertical coordinates, and obtaining the range of the slope k value of each plasmid through least square normal linear fitting; then E-FRET imaging is carried out on the cell sample mixed with the multiple plasmids, background removal, crosstalk correction, region segmentation and region screening are carried out in sequence, and then the cells mixed with the multiple plasmids are distinguished according to the range of the slope k value of each plasmid so as to determine the space standard line of each plasmid; and finally, fitting a plane according to the space standard line to obtain system correction factors G and beta. The system correction factors G and beta determined by the method are accurate and are suitable for different detection systems.

Description

AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof

Technical Field

The invention belongs to the technical field of Fluorescence Resonance Energy Transfer (FRET) detection, and particularly relates to an AutoQT-FRET method based on a correction factor of a primary imaging measurement system and application thereof.

Background

Quantitative Fluorescence Resonance Energy Transfer (FRET) detection is an important technology for studying intermolecular interactions in living cells, analyzing the molecular structure of oligomerized proteins, studying the regulatory mechanisms between proteins in signaling pathways, etc. [ Deal, J., et alT-based sensors of intracellular signals:A biological perspective of the history of FRET."Cellular Signalling(2020):109769.]。Zal[Zal,Tomasz,and Nicholas RJ Gascoigne."Photobleaching-corrected FRET efficiency imaging of live cells."Biophysical journal 86.6(2004):3923-3939.]And the like, a quantitative FRET (E-FRET) measuring method based on 3-cube is provided, and acceptor excitation crosstalk and donor emission crosstalk can be overcome simultaneously. The E-FRET method proceeds through three channels: the combination of the donor detection channel (DD) when excited by donor excitation light, the acceptor detection channel (DA) when excited by donor excitation light, and the acceptor detection channel (AA) when excited by acceptor excitation light eliminates spectral crosstalk, and at the same time, can eliminate spectral crosstalk according to E ═ F_c)/(F_c+G*I_DD) The FRET efficiency E was calculated quantitatively. Wherein I_DDRepresenting the intensity of donor fluorescence detected by the DD channel; g is a system sensitization quenching conversion factor; f_cIs the fluorescence intensity sensitized to the acceptor in the FRET sample.

Accurate measurement of the sensitization-quenching conversion factor G is the key of the quantitative FRET detection technology. Accurate measurement of the correction factor G is therefore particularly important in relation to the intrinsic properties of the instrument and of the fluorescent molecule. In the E-FRET method, zal et al [ Zal, Tomasz, and Nichlas RJ Gascoigne. "Photobleaching-corrected FRET interference of live cells." Biophysical journel 86.6(2004):3923 and 3939.] are measured by Photobleaching with a reference sample identical to the donor and acceptor of the sample to be measured to obtain the sensitization-quenching conversion factor G. However, in the actual measurement, the accurate measurement of the correction factor G is influenced by the defects that the photobleaching method has incomplete photobleaching in the photobleaching process and can only carry out measurement on fixed cells.

Alexis coulomb et al [ Coillomb, Alexis, et al. "QuanTI-FRET. a frame for quantitative FRET measurements in measuring cells." Scientific reports 10.1(2020):1-11 in 2020.]A quantitative triple image FRET method (QuanTi-FRET) is further provided on the basis of the E-FRET method, and a correction factor G and a correction factor beta value related to the concentration ratio of a donor and an acceptor can be simultaneously measured and obtained. The method also obtains an image I related to the fluorescence intensity of the donor and acceptor through the combination of three channels DD, DA and AA_DD、I_DAAnd I_AA. Is subdividedIs distinguished by_DD、I_DAAnd I_AAAnd drawing a spatial scatter diagram for the x axis, the y axis and the z axis, and performing plane least square fitting on the spatial scatter diagram to obtain direction vector absolute values of a spatial plane as correction factors G and beta respectively. The QuanTi-FRET method requires the measurement of at least i (i.gtoreq.2) immobilized plasmids individually. However, the method needs to switch multiple cell samples, is complex to operate, and cannot ensure the same contrast and background signal when imaging the multiple cell samples respectively.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an AutoQT-FRET method based on a correction factor of a primary imaging measurement system, which is a quantitative three-image FRET method further provided on the basis of an E-FRET method.

The invention also aims to provide application of the AutoQT-FRET method based on the correction factor of the primary imaging measurement system.

The purpose of the invention is realized by the following technical scheme:

an AutoQT-FRET method based on a correction factor of a primary imaging measurement system comprises the following steps:

(1) sample preparation

Reference sample: respectively transfecting cells with the I FRET plasmids with different FRET efficiencies, and after the transfected plasmids are successfully expressed, respectively culturing the cells expressing the different FRET plasmids in different sample container carriers until the cells are attached to the walls, and marking as a reference sample 1 and a reference sample 2 … … reference sample i; wherein i is more than or equal to 2 (and is an integer);

② the sample to be tested: respectively transfecting cells with the i FRET plasmids with different FRET efficiencies, merging the cells expressing the different FRET plasmids in the same sample container carrier after the transfected plasmids are successfully expressed, and culturing until the cells adhere to the wall to obtain a sample to be detected; wherein i is more than or equal to 2 (and is an integer);

(2) E-FRET spectroscopic imaging

Thirdly, M fields are selected from the reference sample 1 (the reference sample transfected with the 1 st plasmid) obtained in the step I for E-FRET imaging, and then M cells are selected under the 1 st field to obtainObtaining three channels of average fluorescence intensity I_DD1-1、I_DD1-2……I_DD1-m，I_AA1-1、I_AA1-2……I_AA1-m，I_DA1-1、I_DA1-2……I_DA1-mAnd calculating the acceptor-sensitized fluorescence intensity F_C1-1、F_C1-2……F_C1-m(ii) a Then with I_DD1-1、I_DD1-2……I_DD1-mAs the abscissa, F_C1-1、F_C1-2……F_C1-mA scatter plot is plotted for the ordinate and a least squares linear fit is performed to obtain the slope k at the 1 st field of view of the reference sample 1_1-1(ii) a By analogy, the slope k under the 2 nd to M th field of the reference sample 1 is obtained_1-2、k_1-3……k_1-MAnd determining the maximum value and the minimum value to obtain the slope k of the reference sample 1₁Range of values (k)_{Minimum value}≤k₁≤k_{Maximum value}) (ii) a Wherein M is more than or equal to 10, and M is more than or equal to 10 (both integers);

replacing the reference sample 1 with the reference sample 2, and repeating the step c to obtain the slope k of the reference sample 2₂A range of values; and so on until the slope k of the reference sample i is obtained_iRange of values (denoted as k)₂、k₃……k_i)；

Selecting 1 visual field from the sample to be tested obtained in the step II to carry out E-FRET imaging to obtain three channels I thereof_DD、I_AA、I_DAImage, and the length and width of the area of 100 x 100 pixels are respectively corresponding to I_DD、I_AA、I_DAPerforming region segmentation on the image to obtain n cell region images I_DD-S1、I_DD-S2……I_DD-Sn，I_AA-S1、I_AA-S2……I_AA-Sn，I_DA-S1、I_DA-S2……I_DA-Sn(ii) a Then, starting from the 1 st cell region, the proportion S of the effective data in the cell region is calculated₁And measuring the three-channel average fluorescence intensity of all pixel points in the cell region, wherein the total number of the pixel points is L, and the pixel points are marked as I_DD-1、I_DD-2……I_DD-L，I_AA-1、I_AA-2……I_AA-L，I_DA-1、I_DA-2……I_DA-LAnd simultaneously calculating the receptor-sensitized fluorescence intensity F_C-1、F_C-2……F_C-LThen further calculating to obtain the ratio F of the 1 st cell area_C-1/I_DD-1、F_C-2/I_DD-2……F_C-L/I_DD-LPerforming unimodal Gaussian fitting to obtain unimodal Gaussian function fitting degree value X of the cell region₁And according to I_DD-1、I_DD-2……I_DD-LAnd F_C-1、F_C-2……F_C-LDrawing a scatter diagram, and then obtaining a slope k according to least square normal linear fitting₁' value; by analogy, the proportion S of the effective data in the 2 nd cell area to the nth cell area is calculated in the same way₂、S₃……S_nDegree of fit value X of single-peak Gaussian function₂、X₃……X_nAnd slope k₂′、k₃′……k_n'; wherein n is more than or equal to 25 (the value of n is related to the image size, for example, the 2048 × 2048 pixel picture is divided by 100 × 100, n is 400, the 1024 × 1024 pixel picture is divided by 100 × 100, n is 100, the 512 × 512 pixel picture is divided by 100 × 100, and n is 25);

s degree₁< 30% or X₁(ii) < 90%, removing the cell region if S₁Not less than 30% and X₁More than or equal to 90%, the cell region is retained (excluding the effect on plasmid standard line determination when multiple plasmids are present in a region); in the same way, if S₂< 30% or X₂(ii) < 90%, removing the cell region if S₂Not less than 30% and X₂More than or equal to 90 percent, the cell area is reserved, and the like, R cell areas in total are obtained, and the corresponding slopes are marked as k₁″、k₂″……k_R"; wherein R is more than or equal to 10;

(3) cell classification and determination of plasmid standard lines

Seventhly, obtaining k according to the step (c)₁″、k₂″……k_RSize of "and range of slope values obtained in Steps (c) and (dk₁、k₂……k_iCell classification was performed: if k is₁At k₁In the range of (i), i.e. k₁"corresponding cells are cells transfected with plasmid 1; if k is₁At k₂In the range of (i), i.e. k₁"corresponding cells are cells … … f k transfected with plasmid 2₁At k_iIn the range of (i), i.e. k₁"corresponding cells are cells transfected with the i plasmid; by analogy, respectively combine k₂″、k₃″……k_R"performing classification; then classifying the cells according to the I of each pixel point in all cell regions of each plasmid_DDFc and I_AAA spatial scattergram is drawn for the x, y and z axes, and a spatial straight line is fitted by the least square method to serve as a spatial standard line l for each plasmid₁、l₂……l_i；

(4) Obtaining correction factors for E-FRET systems

Eighty to the step of obtaining the space standard line l of each plasmid₁、l₂……l_iPerforming plane least square fitting to obtain a plane equation Ax + By which is z; wherein A and B are constants, and B is a system correction factor beta₁A/beta is the system correction factor G₁(corresponding to the system correction factors β, G at the 1 st field of view);

ninthly, repeating the steps from five to eight, randomly selecting N different visual fields from the sample to be measured obtained in the step two to carry out E-FRET spectral imaging, and respectively calculating to obtain a system correction factor beta₂、β₃……β_NAnd G₂、G₃……G_NThen, taking the average value of the correction factors to obtain system correction factors G and beta; wherein N is more than or equal to 10.

The types of i in the step (1) are 2-4; preferably 4.

The FRET plasmid in the step (1) is a FRET standard plasmid, and a FRET plasmid with different FRET efficiencies can be constructed by adjusting the distance between a donor and an acceptor (the larger the distance is, the smaller the FRET efficiency is); the efficiency of FRET plasmids can also be measured in advance by methods such as a lifetime measurement method, an E-FRRT measurement method or spectral linear separation and the like (the invention does not need to know the specific efficiency of each plasmid exactly); the FRET plasmid comprises a donor and an acceptor, and the number (concentration) ratio of the donor to the acceptor is 1: n or n: 1 FRET plasmid (standard plasmid of C-V system), n is more than or equal to 1; preferably, the ratio of the number (concentration) of the donor to the number (concentration) of the acceptor is 1: 1, a FRET plasmid; more preferably at least two of C5V, C17V, C32V and CTV.

The sample container carrier in the first step and the second step is a container carrier capable of realizing cell culture and imaging, and comprises but is not limited to a cell culture dish, a pore plate, a glass slide and the like; the invention selects the conventional cell culture dish to culture and image the cells.

The cells in the first step (1) and the second step are preferably Hela cells.

The value range of M in the third step (2) is that M is more than or equal to 20; preferably M is more than or equal to 40; more preferably M.gtoreq.50; the larger the number of cells selected herein, the better.

The selection of the m cells in the third step (2) is preferably realized by the following method: placing a sample container carrier (such as a culture dish) containing cells with transfection plasmids under a wide-field fluorescence microscope, selecting M visual fields, then selecting cells from each visual field, and selecting M cells for measurement; wherein M is more than or equal to 10 (preferably M is more than or equal to 20, more preferably M is more than or equal to 40, and more preferably M is more than or equal to 50), M is more than or equal to 10 (preferably M is more than or equal to 80, more preferably M is more than or equal to 250, and more preferably M is more than or equal to 500); that is, M cells are from M fields of view (all cells in the vector. gtoreq.m cells in all M fields of view).

The number of the selected cells in each visual field is more than 1; preferably 1 to 7; more preferably 1 to 3; more preferably 2 to 3.

The value range of m in the third step (2) is that m is more than or equal to 80; preferably m is more than or equal to 250; more preferably m.gtoreq.500; the larger the number of cells selected herein, the better.

The three-channel average fluorescence intensity in the step (2) is donor fluorescence intensity I detected in the donor fluorescence detection channel when the donor excitation light is excited_DDAcceptor fluorescence detected in the acceptor fluorescence detection channel upon excitation with donor excitation lightLight intensity I_DAAcceptor fluorescence intensity I detected in acceptor fluorescence detection channel when excited by acceptor excitation light_AATo 1, pair_DD、I_DA、I_AAThe original image uses Gaussian filtering to determine the range of the background, and background subtraction and crosstalk correction are sequentially carried out on the original image.

The receptor sensitization fluorescence intensity in the third step (2) is the pair I_DAImage subtraction FRET channel (I)_DA) The fluorescence of the excitation crosstalk of the acceptor and the fluorescence of the emission crosstalk of the donor are corrected to obtain the acceptor-sensitized fluorescence intensity (Fc).

Three channels I described in step (2)_DD、I_AA、I_DAThe image is an image of 2048 × 2048 pixels, 1024 × 1024 pixels, or 512 × 512 pixels; preferably 2048 × 2048 pixels.

The proportion of the effective data in the cell area in the fifth step (2) is calculated by the following method: in the cell region, the background is set to 0 and the non-background is set to 1, and then the proportion of the sum of non-background pixels to the sum of pixels of the entire region is calculated as the proportion of valid data.

The value of R in the step (2) is related to the value of n in the step (2), and the value of n is related to the image size, namely R is more than or equal to 10 and less than or equal to n, namely at least 10 cell areas are ensured to exist, the data of each plasmid can be ensured to exist, and a spatial standard line can be obtained; that is, when the image size is 2048 × 2048 pixels, n is 400, at which time 10 ≦ R ≦ 400; when the image size is 1024 multiplied by 1024 pixels, n is 100, and R is more than or equal to 10 and less than or equal to 100; when the image size is 512 × 512 pixels, n is 25, and at this time, R is 10 ≦ 25.

The cell classification in step (3) can also be achieved by:

(a) selecting N 'cell regions under N' visual fields for E-FRET imaging on the sample to be detected obtained in the step II, and obtaining three-channel average fluorescence intensity I pixel by pixel_DDˊ、I_DAˊ、I_AAAnd calculating the intensity of receptor-sensitized fluorescence F thereof_CAnd with I of each pixel_DDˊ、F_Cˊ、I_AAIs emptyAn inter-coordinate point;

(b) respectively calculating the 1 st spatial coordinate point to the spatial standard line l obtained in the step (c)₁、l₂……l_iDistance d of₁、d₂……d_i(ii) a Then d is put₁、d₂……d_iSorting by magnitude of value, if d₁And if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l₁Namely, the cell region corresponding to the space coordinate point belongs to the cell transfected with the FRET plasmid of the 1 st type; in the same way, if d₂And if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l₂That is, the region of the cell corresponding to the spatial coordinate point belongs to the region d of the cell … … transfected with the FRET plasmid of the 2 nd species_iAnd if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l_iNamely, the cell region corresponding to the space coordinate point belongs to the cells transfected with the i-th FRET plasmid; and by analogy, classifying the spatial coordinate point corresponding to each pixel to realize the classification of the cells (plasmids).

The preferable value range of N in the step (5) is as follows: n is more than or equal to 15; more preferably, N.gtoreq.40.

The AutoQT-FRET method based on the correction factor of the primary imaging measurement system is applied to the correction factor of the measurement system.

The system correction factors are system correction factors G and beta.

The basic principle of the invention is as follows:

(1) for FRET samples imaged by a three-channel method, the fluorescence intensity I of the three channels for the acceptor is obtained by imaging_DD、I_DA、I_AA. It can be written as a function of photophysical and instrumental parameters, the number n of donors in the pixel under consideration^DAnd the number of acceptors n^AFluorophore and FRET probability (efficiency) E:

I_AA＝n^A*L_A*σ^A _Aex*Φ_A*η^Aem _Adet (1)

I_DD＝n^D*L_D*σ^D _Dex*(1-E)*Φ_D*η^Dem _Ddet (2)

I_DA＝n^D*L_D*σ^D _Dex*E*Φ_A*η^Aem _Adet+n^D*L_D*σ^D _Dex*(1-E)*Φ_D*η^Dem _Adet+n^A*L_D*σ^A _Dex*Φ_A*η^Aem _Adet (3)

wherein L is_AExcitation intensity at a wavelength selected to excite fluorescent molecule a; l is_DExcitation intensity at a wavelength selected to excite fluorescent molecule D; sigma^A _AexIs the absorption cross section of A at the excitation wavelength of A; sigma^D _DexIs the absorption cross section of D at the excitation wavelength of D; sigma^A _DexIs the absorption cross section of D at the excitation wavelength of A; phi_AQuantum yield of a; phi_DQuantum yield of D; eta^Aem _AdetA detection efficiency for emitting photons in detection channel a; eta^Dem _DdetA detection efficiency for emitting photons in detection channel D; eta^Dem _AdetThe detection efficiency of a for emitting photons in the detection channel D.

To simplify the operation, four correction factors are defined by equation (4) to simplify equations (1) - (3): the direct excitation correction factor is a; the leakage correction factor is d; the correction factor for different detection efficiencies in the two channels is G; the correction factor for the different excitation efficiencies in the two channels is β.

(2) By deducting FRET channel (I)_DA) Fluorescence of excitation crosstalk of intermediate acceptor and fluorescence of emission crosstalk of donor to obtain fluorescence intensity F sensitized to acceptor in FRET sample_c：

Fc＝I_DA-aI_AA-dI_DD (5)

Thus FRET efficiency can be expressed as:

(3) meanwhile, the stoichiometric ratio is defined as the relative number of donor molecules relative to the total number of fluorophores in each pixel:

from the equations (1) (2), n can be derived^DAnd n^AAnd inserted into equation (7). By simplifying with the use of the correction factor β defined in equation (4), equation (7) reduces to:

(4) to accurately obtain the correction factors G and β, equation (8) is rewritten as:

this is represented by { I_DD、Fc、I_AAThe equation of the plane in the defined three-dimensional space. If the stoichiometric ratio S is known, the experimental data { I_DD、Fc、I_AAThe correction factors G and β are determined by least squares fitting to a plane.

(5) Multiple sets of { I } sets can be obtained by measuring 2 or more samples of FRET tandem structure containing 1 donor and 1 acceptor (number ratio 1: 1)_DD、Fc、I_AAAnd through the pair { I }_DD、Fc、I_AAAnd performing least square fitting on a space plane, wherein absolute values of space vectors of the fitting plane are correction factors G and beta in the formula (9) respectively.

(6) K value range for cells transfected with FRET-derived plasmids using single-transfer plasmid samples: the correction factor G is only related to the instrumentAnd the intrinsic properties of the fluorescent molecule, whereas the FRET efficiency E is associated with a different donor FRET pair. Therefore, I shown in the following formula (10)_DDSlope of Fc

Showing tandem FRET plasmids having different FRET efficiencies.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention belongs to the technical field of FRET quantitative detection, and the method utilizes a cell sample (the sample contains 2 or more than 2 FRET tandem plasmids with the ratio of donor to acceptor being 1: 1) to simultaneously measure a system correction factor G (system sensitization quenching conversion factor) and beta (excitation scale factor of two channels), and specifically comprises the following steps: obtaining the k value range of each plasmid by using E-FRET data of single-transfer plasmid cells; sequentially carrying out processes of background removal, crosstalk correction, region segmentation, region screening, determination of a space standard line of each plasmid and the like on cell E-FRET image data mixed with various plasmids; fitting a plane according to the determined space standard line of each plasmid and obtaining a system correction factor; and the spatial standard lines can be used to classify one dish of images of multiple granulocytes.

(2) The invention provides an AutoQT-FRET method based on a correction factor of a primary imaging measurement system, thereby improving the intelligence and the accuracy of correction parameters G and beta of the measurement system and being the basis for developing an intelligent FRET correction system.

(3) The G and the beta obtained by the method are more accurate and are suitable for different detection systems, so that the method can greatly promote the application range of the (AutoQT-FRET) method, and further improve the application range of the FRET detection technology in cell biology.

(4) Compared with the traditional method, the method can finish the measurement of the correction factor only by one cell sample (each plasmid is not required to be separately measured, and the problems of different contrast and background signals exist in the separate measurement), and in addition, the method can be used for measuring and calculating the correction factor on line (for example, data is processed by a computer), so that the use threshold is reduced, and the efficiency is improved.

Drawings

FIG. 1 is a diagram showing the procedure and results of determining the range of different plasmids k according to the present invention; wherein, (a) is DA channel image for E-FRET spectral imaging of cell sample of single-transfer four plasmids; (b) to select the cell region encircled by the red box in (a), the cell region I is subjected to_DDDrawing a scatter diagram by the Fc data, and fitting by a least square method to obtain a linear graph of the slope k of the cell region; (c) to perform a statistical analysis of the k-values of a total of 265 cells in 40 fields, a statistical map of the range of slope k-values for different plasmids was obtained.

FIG. 2 is a graph showing the process and results of determining spatial standard lines of different plasmids according to the present invention; wherein, (a) a petri dish is transfected with E-FRET spectroscopic imaging data of any one field of four different plasmid cell samples; (b) to select the cell region circled by the red box in (a), the cell region was subjected to pixel-by-pixel Fc/I_DDCounting the frequency distribution of the values and obtaining a fitting graph by single-peak Gaussian fitting; (c) results are presented as spatial standard lines for three plasmids C5V, C17V, C32V, CTV in this field; (d) a visual field data classification template map was prepared based on (a) the distribution of different plasmids in the visual field (in the figure: blue is data of cell-transfected C5V plasmid, red is data of cell-transfected C17V plasmid, yellow is data of cell-transfected C32V plasmid, and purple is data of cell-transfected CTV plasmid).

FIG. 3 is a graph of the statistical results of correction factors obtained using different methods.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

Example 1

1. Plasmids

Given donor-acceptor pairs: the donor is gene coding fluorescent protein Cerulean (short for C), and the acceptor is gene coding fluorescent protein Venus (short for V);

FRET tandem plasmid structure C5V: a FRET tandem plasmid structure consisting of a 5 amino acid linker linking C and V;

FRET tandem plasmid structure C17V: a FRET tandem plasmid structure consisting of a 17 amino acid linker linking C and V;

FRET tandem plasmid structure C32V: a FRET tandem plasmid structure consisting of a 32 amino acid linker linking C and V;

FRET tandem plasmid structure CTV (Cerulean-TRAF-Venus): a FRET tandem plasmid structure consisting of a 229 amino acid linker linking C and V; wherein TRAF is a receptor-associated factor domain comprising a long chain tumor necrosis factor of 229 amino acids;

the source is as follows: the donor plasmids Cerulean (C), the acceptor plasmid Venus (V) and the FRET reference plasmids C5V, C17V, C32V and CTV were purchased from the Addge plasmid library, USA.

2. Wide-field spectral microscopic imaging system

Wide field fluorescence microscopes were manufactured by Olympus, Japan, under model IX 73. The light source was a mercury lamp of the olympus HGLGPS series, japan. The objective lens is an oil lens with the magnification of 40 and the numerical aperture of 1.3(40 multiplied by 1.3NA), one exciting rotating wheel provided with four exciting sheets, one rotating wheel provided with eight cubes (each cube can be provided with one exciting sheet, one splitting sheet and one emitting sheet respectively), one electric emitting rotating wheel provided with six emitting sheets, and the electric emitting rotating wheel is externally connected with a CCD camera. The wavelength of the excitation light is selected by rotating the excitation wheel.

3. Cell culture and plasmid transfection

Hela cells were obtained from university of river, Guangzhou, China, and 10% (v/v) of newborn bovine serum was added to DMEM medium and cultured in an incubator at 37 ℃ containing 5% (v/v) carbon dioxide. Trypsin for cellsDigesting, transferring to a cell culture dish, culturing for 24 hours, and then using an in vitro transfection reagent Turbofect when the cells grow to 70-90 percent^TMThe plasmid was transiently transferred into cells. The specific steps of transfection are as follows:

(1) taking a sterilized EP tube, firstly adding 100 mu L of serum-free DMEM medium, then adding 1-2 mu L of transfection reagent, then adding 1-2 mu L (500-600 ng/mu L) of plasmid, gently blowing and beating for 6-8 times, and then standing for 20 minutes;

(2) after 20 minutes, adding 100 mu L of serum-free DMEM medium into the EP tube, and gently mixing;

(3) washing cells in the culture dish for 2-3 times by using a serum-free DMEM medium or PBS (phosphate buffered saline), mainly washing off dirt such as dead cells and the like, then transferring the mixture in the step (2) into the culture dish, and putting the culture dish back into the culture box for 4-6 hours;

(4) (ii) for samples of single transfer plasmids C5V, C17V, C32V and CTV in a culture dish, E-FRET imaging can be respectively carried out after the steps (1) to (3) are completed, and donor fluorescence intensity I detected in a donor fluorescence detection channel when excited by donor excitation light is obtained_DDIntensity of acceptor fluorescence I detected in acceptor fluorescence detection channel upon excitation with donor excitation light_DAAnd the intensity of acceptor fluorescence I detected in the acceptor fluorescence detection channel when excited by the acceptor excitation light_AA(i.e., three-channel mean fluorescence intensity I_DD、I_DA、I_AA) And calculating the acceptor-sensitized fluorescence intensity F_C(ii) a Wherein, F_CTo subtract FRET channel (I)_DA) Fluorescence intensity sensitized to acceptor in FRET sample obtained by fluorescence of excitation crosstalk of acceptor and fluorescence of emission crosstalk of donor_c。

② for a sample containing the multi-plasmid (C5V, C17V, C32V and CTV) in the cells of a culture dish, the preparation method comprises the following steps: after the cells independently transfecting plasmids are subjected to step (3) for 4-6 hours respectively, absorbing transfection liquid, cleaning the cells in the culture dish for 2-3 times by using serum-free DMEM culture medium or PBS, adding 100 mu L of pancreatin cell digestive fluid into the culture dish to digest the cells transfected with different plasmids in different culture dishes, absorbing the digestive fluid, slightly blowing the cells for 2-3 times by using 100 mu L of serum-free DMEM culture medium, putting cell turbid liquid blown from different culture dishes into the same EP tube, uniformly mixing for 2-3 times, and putting the cell turbid liquid into a new culture dish again, wherein the ratio of the DMEM culture medium to fetal bovine serum is 10: 1 (volume ratio), adding fetal bovine serum, culturing for 8-12 hours, and allowing the cells to adhere to the wall for experiment.

4. Multi-plasmid cotransfer sample imaging process

CTV, C5V, C17V and C32V were transfected into Hela cells separately according to the plasmid transfection procedure described above, and then pooled into one dish for expression maturation (cell attachment) followed by E-FRET spectroscopy. The wide field fluorescence microscope was configured as follows: light of 436/20nm and 470/20nm is used as exciting light of Cerulean and Venus respectively, and channels of 510nm and 530nm are used as detection channels of Cerulean and Venus fluorescence respectively. Under 436nm excitation, the detector collects images of all detection channels, and under 470nm excitation, the detector collects images of only 530nm detection channels, and 3 images are recorded as E-FRET data of one field of view.

5. Data processing

5.1 determining the k value range of FRET plasmid transfected cells according to E-FRET data of a multi-plasmid single-transfer sample, and the specific process is as follows:

in the step (4), 40 fields (10 fields for each plasmid sample) are randomly selected from the multi-plasmid single-transfer sample, then cells are selected in each field, and 265 cells are selected for measurement in the experiment. Obtaining an image I after background subtraction and crosstalk correction according to E-FRET spectral imaging_DD、Fc、I_AA. Drawing I pixel by pixel for each field of view_DDFc dot plot (as I)_DDAbscissa and Fc ordinate), and performing least square fitting to calculate a slope k value of each field, which is as follows:

as shown in FIG. 1(a), for the FRET reference plasmid C5V, 1 field was selected for E-FRET spectroscopic imaging, and then the cell region enclosed by the red box in FIG. 1(a) was selected (i.e., the field was selected first, and then the cell region was selected under the field, which has no special requirement and is free to be selectedSelecting and controlling the size of the selected cell region, such as whole cells or part of cells, wherein the more data, the more accurate the measurement, and 265 cells are selected in total), and obtaining the three-channel average fluorescence intensity I of each cell region respectively_DD、I_DA、I_AAAnd calculating the acceptor-sensitized fluorescence intensity F according to the calculated result_C. Then based on I of the selected cell region_DDDrawing a scatter diagram by Fc data, and fitting by a least square method to obtain a diagram of a slope k fitting straight line of the cell region, wherein the diagram is shown in a figure 1 (b); by analogy, E-FRET spectroscopic imaging was performed on cell samples of the single-transfer FRET reference plasmids C5V, C17V and C32V, and then slope k was calculated by fitting all cell regions in 10 fields of each plasmid. The slope k values of all cell regions were statistically classified according to different plasmids, resulting in the range of slope k values for different plasmids, as shown in FIG. 1 (c). The finally determined ranges of the slope k values of the different plasmids are respectively: C5V: k is more than or equal to 5.5_C5V≤8.5；C17V：3.2≤k_C17V≤5；C32V：2≤k_C32V≤3；CTV：0.2≤k_CTV≤1.3。

TABLE 1 slope k value ranges for different plasmids

Species of plasmid	C5V	C17V	C32V	CTV
					Slope k	[5.5,8.5]	[3.2,5]	[2,3]	[0.2,1.3]

Here, the measurement can be performed on Hela cells transfected with CTV, C5V, C17V and C32V individually (different dishes), and the cells can be selected individually (the number of cells to be selected can be selected as the case may be, preferably 10 or more fields (preferably 20 or more) are selected for each dish transfected with a different plasmid, and 1 or more cells (preferably 3 to 7 cells) are selected for each field). The experiment chose to measure on different dishes, i.e. directly on single-transfer plasmid samples in step 4.

5.2 determining the spatial standard line for each plasmid based on E-FRET data from multiple plasmid cotransformation samples:

5.2.1 Classification of plasmids according to the slope k values and determination of the spatial Standard line for each plasmid

For a total of 223 cells in 40 visual fields of the sample (containing C5V, C17V, C32V and CTV) co-transformed by multiple plasmids in the step 3, calculating a slope k value pixel by pixel for each cell region, classifying according to k value ranges of 4 different plasmids determined in the step 5.1, and then performing space straight line least square fitting to obtain standard lines of different plasmids, wherein the specific steps are as follows:

as shown in fig. 2(a), after performing E-FRET spectral imaging on a cell sample co-transformed with four plasmids, original images DD and AA are obtained; obtaining I after background deduction and crosstalk correction_DD、I_DA、I_AAAn image; according to the area length and width I of 100 x 100 pixels_DD、I_DA、I_AAAnd carrying out region segmentation on the image. Selecting the cell region (i.e. 100 x 100 pixel region) encircled by red box in fig. 2(a), calculating the proportion of effective data in the region (in the region, the background is already set to 0 and the non-background is set to 1 in the background subtraction process, so that the proportion of the sum of non-background pixels to the sum of pixels in the whole region is the effective data proportion) pixel by pixel, namely S_-10.86. The cell region was then subjected to pixel-by-pixel Fc/I_DDOf valueFrequency distribution statistics (i.e. for the first selected cell, in this cell region, starting from the 1 st pixel point, obtaining E-FRET data Fc and I thereof_DDThen calculating Fc/I_DDA value; then obtaining E-FRET data Fc and I of the 2 nd pixel point_DDThen it calculates Fc/I_DDThe value … … is analogized until the last pixel point of the cell region (the size of the map used in the present invention is 2048 × 2048 pixels (pixels)); a unimodal gaussian fit was performed, see fig. 2(b), to obtain the degree of fitting of the unimodal gaussian function for this cell region, i.e., X ═ 0.48 (gaussian fit was performed using matlab software on the blue points of fig. 2(b) obtained statistically, to obtain the gaussian function (red line)). Finally, the slope k value of the least square fitting of the cell region is calculated to be 5.23, and the specific calculation method is the same as the step 5.1 (corresponding to I through each pixel point in the cell region)_DDAnd drawing a scatter diagram by the Fc data, and fitting a straight line to obtain a slope k value).

Note: the above is based on Aoyin et al [ Yin, Ao, et al ] "Measuring calibration factors by imaging a dis of cell expression measurements" Cytometry Part A (2021).]The Fc/I of the same plasmid is proposed_DDThe frequency histogram should satisfy a monomodal gaussian distribution, and Fc/IDD frequency histograms of two or more plasmids show the presence of multiple gaussian peaks corresponding to the number of plasmids. According to the principle, the Fc/I is calculated region by region for the regions screened out in the step_DDFrequency distribution and fitting with a unimodal gaussian function.

Obtaining effective data proportion S of all cell areas in the visual field, Gaussian function fitting degree X and slope k according to the same method, and respectively recording the effective data proportion S, the Gaussian function fitting degree X and the slope k as S_-1、S_-2……S_-223，X_-1、X_-2……X_-223，k_-1、k_-2……k_-223. And respectively judging whether the effective data proportion in each region exceeds 30%, and if the effective data proportion in each region is less than 30%, removing the cell region. If the cell area is larger than or equal to 30%, the cell area is reserved; and then judging whether the fitting degree of the Gaussian function in each remaining cell area is greater than or equal to 90%, and if the fitting degree of the Gaussian function in each remaining cell area is less than 90%, removing the cell area. If greater thanEqual to 90% the cell area is retained; finally, the slope k values of the remaining cell regions are determined according to the k value range (k is more than or equal to 5.5) of different plasmids determined in the step 5.1_C5V≤8.5；3.2≤k_C17V≤5；2≤k_C32V≤3；0.2≤k_CTV≦ 1.3) for classification (the k-value range is determined by the maximum and minimum values of the valid data measured for each plasmid, with a small amount of data error possible, e.g. data with k-values outside the above ranges are obtained, which can be re-measured or removed), i.e. the remaining cell area is divided to determine which plasmid (C5V, C17V, C32V or CTV) it belongs to. Then, the classified cell region set is subjected to fitting of a space straight line least square method (namely I corresponding to the classified cell region_DDFc and I_AADrawing a spatial scatter diagram for x, y and z axes, and fitting) to obtain spatial standard lines l of different plasmids₁、l₂、l₃、l₄See fig. 2 (c).

5.2.2 Classification of visual field data by spatial Standard lines of different plasmids

In addition to the above classification of plasmids using slope k values, the four spatial standard lines l of plasmids obtained in step 5.2.1 can be used₁、l₂、l₃、l₄(corresponding to 4 kinds of plasmids) and the formula (11), the visual field data of FIG. 2(a) was classified, and a template map for classification of the visual field data was prepared:

wherein

Is a standard line L_i(i is 1,2 … … n) and point M is a straight line L_iAt any point, point M₀Is the coordinates of the spatial point sought.

Firstly, as shown in FIG. 2(a), E-FRET spectral imaging is carried out on a cell sample co-transformed with four plasmids to obtain original images DD, DA and AA, and background subtraction and subtraction are carried outAfter crosstalk correction, I is obtained_DD、Fc、I_AA(ii) a Then with I_DD、Fc、I_AACalculating the coordinate of a space point P corresponding to the 1 st pixel point according to the formula (1) for x, y and z axes as { I }_DD、Fc、I_AATo the space standard line l₁Distance d of₁；

Secondly, according to the same method, calculating to obtain a space point P { I_DD、Fc、I_AATo the space standard line l₂Distance d of₂Space point P { I_DD、Fc、I_AATo the space standard line l₃Distance d of₃And spatial point P { I_DD、Fc、I_AATo the space standard line l₃Distance d of₄；

Comparison d₁、d₂、d₃Size, the spatial point P { I }_DD、Fc、I_AAClassify as a spatial standard line of closest distance, i.e. d₁At the minimum, the spatial point P is classified as a spatial standard line l₁In the same way, e.g. d₂At the minimum, the spatial point P is classified as a spatial standard line l₂E.g. d₃At the minimum, the spatial point P is classified as a spatial standard line l₃E.g. d₄At the minimum, the spatial point P is classified as a spatial standard line l₄；

And fourthly, classifying all the pixel points in the view to obtain four data set image templates of C5V, C17V, C32V and CTV (namely, marking the background in the view as 0, marking the pixel point classified as C5V as 1, marking the pixel point classified as C17V as 2, marking the pixel point classified as C32V as 3 and marking the pixel point classified as CTV as 4). The four datasets were merged (simple matrix addition, each dataset being an n x n size image; where n is the original image size 1024 x 1024 or 512 x 512) and the visual field data sort template map was made with different color labels, see fig. 2 (d): blue is data of cells transfected with C5V plasmid, red is data of cells transfected with C17V plasmid, yellow is data of cells transfected with C32V plasmid, and purple is data of cells transfected with CTV plasmid.

5.3 calculate the system correction factors G and β:

four plasmid spatial standard lines l obtained in step 5.2 were used₁、l₂、l₃、l₄And equation (10), calculating system correction factors G and β; for the four plasmid space standard lines l obtained in step 5.2₁、l₂、l₃、l₄And performing least square fitting on the space plane, wherein the fitting plane equation should satisfy the form of Ax + By ═ z (the distance from each straight line to the plane is calculated, and the four straight line-to-plane distances and the plane with the minimum distance are the fitting plane). The visual field data is fitted to obtain a plane equation of 2.01I_DD+0.59Fc＝I_AAAccording to formula (10), wherein G β is 2.01, β is 0.59; the fit therefore gives a G value of 3.70 for this field.

5.4 authentication

In order to test whether the data of the correction factors obtained by the method is accurate, the data of 200 cells in 22 fields selected by a co-transformed sample of various plasmids are analyzed by using an AutoQT-FRET method, a two-hybrid-multi-plasmid method and a TP-G method, and the specific steps are as follows:

(1) according to the two-hybrid-multi-plasmid method (reference: Butz, E.S., et al. "Quantifying macromolecular interactions in living cells using FRET two-hybrid assays," Nature Protocols 11.12(2016):2470.), the template image matrixes of the four plasmid datasets C5V, C17V, C32V and CTV classified in step 5.2.2 are respectively multiplied by the template image matrixes of the plasmid datasets obtained in step 5.2.2 to obtain I after background subtraction and crosstalk correction_DD、Fc、I_AAImage matrix, obtaining I of each plasmid_DD、Fc、I_AAAnd (5) an image matrix. And determining each plasmid I_DD、Fc、I_AAAverage value of image matrix, denoted as I_DD(C5V)、Fc(C5V)、I_AA(C5V)，I_DD(C17V)、Fc(C17V)、I_AA(C17V)，I_DD(C32V)、Fc(C32V)、I_AA(C32V), and I_DD(CTV)、Fc(CTV)、I_AA(CTV). The point is then coordinated as

The four points are drawn in a plane rectangular coordinate system, and the G value obtained according to a two-hybrid-multi-plasmid method in the visual field is obtained by performing least square fitting on the four points. Averaging the G values obtained from 22 visual fields to obtain a correction factor G value of 3.72 +/-0.93 (FIG. 3);

(2) fitting the same data set using the AutoQT-FRET method of the invention (see steps 5.1 to 5.3 for a specific method) resulted in a mean correction factor of 3.51 ± 0.81 for 22 fields (fig. 3).

(3) According to the TP-G method (reference: Chen H, Puhl H L, Koushik S V, et al. measurement of FRET efficiency and ratio of don to acceptor con-centration in living cells [ J)]Biophysical journal,2006,91(5): L39-L41.) also gave values for the correction factor G. The TP-G method obtains the G value by a double-plasmid method, so that the G value can be calculated by combining two plasmids in a system of co-transforming four plasmids. We named the method of obtaining the correction factor G by C5V and C17V plasmids (in step 5.2.2, the four plasmids in the visual field of the cotransformation plasmid sample have been distinguished by C5V, C17V, C32V and CTV, so the correction factor G can be obtained by pairing two plasmids and calculating) as TP-G-1 method, the method of obtaining the correction factor G by C17V and C32V plasmids as TP-G-2 method, and the method of obtaining the correction factor G by C32V and CTV plasmids as TP-G-3 method. Similarly, the template image matrixes of the four plasmid data sets of C5V, C17V, C32V and CTV classified in the step 5.2.2 are respectively subjected to point multiplication by the template image matrixes of the four plasmid data sets obtained in the step 5.2, and subjected to background subtraction and crosstalk correction to obtain I_DD、Fc、I_AAImage matrix, obtaining I of each plasmid_DD、Fc、I_AAAnd (5) an image matrix. And determining each plasmid I_DD、Fc、I_AAAverage value of image matrix, denoted as I_DD(C5V)、Fc(C5V)、I_AA(C5V)，I_DD(C17V)、Fc(C17V)、I_AA(C17V)，I_DD(C32V)、Fc(C32V)、I_AA(C32V), and I_DD(CTV)、Fc(CTV)、I_AA(CTV). According to equation (12):

wherein 1 and 2 represent two different plasmids. Calculating the mean value of the correction factors to be 5.42 +/-1.81 by a method of obtaining 22 visual fields TP-G-1 according to the formula (12); the mean value of correction factors calculated by a TP-G-2 method is 4.23 +/-1.64; the mean correction factor calculated by the TP-G-3 method was 2.98. + -. 1.33 (FIG. 3).

The error of the correction factor G obtained by comparing the two-hybrid method and the AutoQT-FRET method is 5.64 percent and is within the error allowable range. Therefore, compared with the double-hybridization method, the correction factor obtained by the AutoQT-FRET method has stability. The time and workload for solving the correction factor G can be greatly reduced by using the AutoQT-FRET method.

5.5 the above experiment is to fit the spatial standard line of the visual field in any visual field, and finally obtain the correction factor of the visual field. In order to enable the obtained data of the correction factors to be more accurate, the data can be measured for multiple times and then averaged, for example, multiple visual fields (at least 10-15 visual fields) are selected, G and beta under different visual fields are obtained according to the methods of 5.2 and 5.3, and then the average values are obtained to obtain the correction factors G and beta of the automatic QT-FRET system.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. An AutoQT-FRET method based on a correction factor of a primary imaging measurement system is characterized by comprising the following steps:

(1) sample preparation

Reference sample: respectively transfecting cells with the I FRET plasmids with different FRET efficiencies, and after the transfected plasmids are successfully expressed, respectively culturing the cells expressing the different FRET plasmids in different sample container carriers until the cells are attached to the walls, and marking as a reference sample 1 and a reference sample 2 … … reference sample i; wherein i is more than or equal to 2;

② the sample to be tested: respectively transfecting cells with the i FRET plasmids with different FRET efficiencies, merging the cells expressing the different FRET plasmids in the same sample container carrier after the transfected plasmids are successfully expressed, and culturing until the cells adhere to the wall to obtain a sample to be detected; wherein i is more than or equal to 2;

(2) E-FRET spectroscopic imaging

Thirdly, M visual fields are selected from the reference sample 1 obtained in the step I to carry out E-FRET imaging, and then M cells are selected under the 1 st visual field to obtain three-channel average fluorescence intensity I_DD1-1、I_DD1-2……I_DD1-m，I_AA1-1、I_AA1-2……I_AA1-m，I_DA1-1、I_DA1-2……I_DA1-mAnd calculating the acceptor-sensitized fluorescence intensity F_C1-1、F_C1-2……F_C1-m(ii) a Then with I_DD1-1、I_DD1-2……I_DD1-mAs the abscissa, F_C1-1、F_C1-2……F_C1-mA scatter plot is plotted for the ordinate and a least squares linear fit is performed to obtain the slope k at the 1 st field of view of the reference sample 1_1-1(ii) a By analogy, the slope k under the 2 nd to M th field of the reference sample 1 is obtained_1-2、k_1-3……k_1-MAnd determining the maximum value and the minimum value to obtain the slope k of the reference sample 1₁Range of values: k is a radical of_{Minimum value}≤k₁≤k_{Maximum value}(ii) a Wherein M is more than or equal to 10, and M is more than or equal to 10;

replacing the reference sample 1 with the reference sample 2, and repeating the step c to obtain the slope k of the reference sample 2₂A range of values; and so on until the slope k of the reference sample i is obtained_iA range of values;

selecting 1 visual field from the sample to be tested obtained in the step II to carry out E-FRET imaging to obtain three channels I thereof_DD、I_AA、I_DAImage, and the length and width of the area of 100 x 100 pixels are respectively corresponding to I_DD、I_AA、I_DAPerforming region segmentation on the image to obtain n cell region images I_DD-S1、I_DD-S2……I_DD-Sn，I_AA-S1、I_AA-S2……I_AA-Sn，I_DA-S1、I_DA-S2……I_DA-Sn(ii) a Then, starting from the 1 st cell region, the proportion S of the effective data in the cell region is calculated₁And measuring the three-channel average fluorescence intensity of all pixel points in the cell region, wherein the total number of the pixel points is L, and the pixel points are marked as I_DD-1、I_DD-2……I_DD-L，I_AA-1、I_AA-2……I_AA-L，I_DA-1、I_DA-2……I_DA-LAnd simultaneously calculating the receptor-sensitized fluorescence intensity F_C-1、F_C-2……F_C-LThen further calculating to obtain the ratio F of the 1 st cell area_C-1/I_DD-1、F_C-2/I_DD-2……F_C-L/I_DD-LPerforming unimodal Gaussian fitting to obtain unimodal Gaussian function fitting degree value X of the cell region₁And according to I_DD-1、I_DD-2……I_DD-LAnd F_C-1、F_C-2……F_C-LDrawing a scatter diagram, and then obtaining a slope k according to least square normal linear fitting₁' value; by analogy, the proportion S of the effective data in the 2 nd cell area to the nth cell area is calculated in the same way₂、S₃……S_nDegree of fit value X of single-peak Gaussian function₂、X₃……X_nAnd slope k₂′、k₃′……k_n'; wherein n is more than or equal to 25;

s degree₁< 30% or X₁(ii) < 90%, removing the cell region if S₁Not less than 30% and X₁The cell area is reserved if the cell area is more than or equal to 90 percent; in the same way, if S₂< 30% or X₂(ii) < 90%, removing the cell region if S₂Not less than 30% and X₂More than or equal to 90 percent, the cell area is reserved, and the like, R cell areas in total are obtained, and the corresponding slopes are marked as k₁″、k₂″……k_R″；

Wherein R is more than or equal to 10;

(3) cell classification and determination of plasmid standard lines

Seventhly, obtaining k according to the step (c)₁″、k₂″……k_R"size and range k of slope values obtained in Steps (c) and (d)₁、k₂……k_iCell classification was performed: if k is₁At k₁In the range of (i), i.e. k₁"corresponding cells are cells transfected with plasmid 1; if k is₁At k₂In the range of (i), i.e. k₁"corresponding cells are cells … … f k transfected with plasmid 2₁At k_iIn the range of (i), i.e. k₁"corresponding cells are cells transfected with the i plasmid; by analogy, respectively combine k₂″、k₃″……k_R"performing classification; then classifying the cells according to the I of each pixel point in all cell regions of each plasmid_DDFc and I_AAA spatial scattergram is drawn for the x, y and z axes, and a spatial straight line is fitted by the least square method to serve as a spatial standard line l for each plasmid₁、l₂……l_i；

(4) Obtaining correction factors for E-FRET systems

Eighty to the step of obtaining the space standard line l of each plasmid₁、l₂……l_iPerforming plane least square fitting to obtain a plane equation Ax + By which is z; wherein A and B are constants, and B is a system correction factor beta₁A/beta is the system correction factor G₁；

2. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

the cell classification in step (3) can also be achieved by:

(a) selecting N under N' visual fields for the sample to be detected obtained in the step IIE-FRET imaging is carried out on the cell region of the' cell, and three channels of average fluorescence intensity I are obtained pixel by pixel_DDˊ、I_DAˊ、I_AAAnd calculating the intensity of receptor-sensitized fluorescence F thereof_CAnd with I of each pixel_DDˊ、F_Cˊ、I_AA' is a spatial coordinate point;

(b) respectively calculating the 1 st spatial coordinate point to the spatial standard line l obtained in the step (c)₁、l₂……l_iDistance d of₁、d₂……d_i(ii) a Then d is put₁、d₂……d_iSorting by magnitude of value, if d₁And if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l₁Namely, the cell region corresponding to the space coordinate point belongs to the cell transfected with the FRET plasmid of the 1 st type; in the same way, if d₂And if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l₂That is, the region of the cell corresponding to the spatial coordinate point belongs to the region d of the cell … … transfected with the FRET plasmid of the 2 nd species_iAnd if the minimum value is reached, classifying the spatial coordinate point as a spatial standard line l_iNamely, the cell region corresponding to the space coordinate point belongs to the cells transfected with the i-th FRET plasmid; and by analogy, classifying the spatial coordinate point corresponding to each pixel to realize the classification of the cells.

3. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

the types of i in the step (1) are 2-4;

the number ratio of the FRET plasmid as a donor to an acceptor in the (1) part is 1: n or n: 1, n is more than or equal to 1;

the value range of M in the third step (2) is that M is more than or equal to 20;

the value range of m in the third step (2) is that m is more than or equal to 80;

the value range of R in the step (2) is more than or equal to 10 and less than or equal to 400;

the numerical range of N in the step (5) is as follows: n is more than or equal to 15.

4. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 3, characterized in that:

the number ratio of the FRET plasmid as a donor to an acceptor in the (1) part is 1: 1, a FRET plasmid;

the value range of M in the third step (2) is that M is more than or equal to 40;

the value range of m in the third step (2) is that m is more than or equal to 250;

the numerical range of N in the step (5) is as follows: n is more than or equal to 40.

5. The AutoQT-FRET method based on primary imaging measurement system correction factors of claim 4, characterized in that:

the FRET plasmid in the step (1) is at least two of C5V, C17V, C32V and CTV;

the value range of M in the third step (2) is that M is more than or equal to 50;

the value range of m in the third step (2) is that m is more than or equal to 500.

6. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

7. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

the cells in the first step and the second step are Hela cells.

8. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

the selection of the m cells in the third step (2) is realized by the following mode: placing a sample container carrier filled with cells of transfection plasmids under a wide-field fluorescence microscope, firstly selecting M visual fields, then selecting cells from each visual field, and selecting M cells for measurement; wherein M is more than or equal to 10, and M is more than or equal to 10;

the number of the selected cells in each field of view is 1 or more.

9. The AutoQT-FRET method based on primary imaging measurement system correction factors according to claim 1, characterized in that:

the sample container carrier in the first step and the second step is a cell culture dish, a pore plate or a glass slide;

three channels I described in step (2)_DD、I_AA、I_DAThe image is an image of 2048 × 2048 pixels, 1024 × 1024 pixels, or 512 × 512 pixels.

10. The application of the AutoQT-FRET method based on the primary imaging measurement system correction factor in the measurement system correction factor in any one of claims 1 to 9 is characterized in that:

the system correction factors are system correction factors G and beta.