CN113049555B - Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application - Google Patents
Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application Download PDFInfo
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Abstract
The invention discloses a method for measuring a correction factor of an excited emission spectrum linear separation quantitative FRET system based on a cell sample of the same system and application thereof. The method comprises the following steps: in the same system (such as a dish of cells), the weight factor graph W is obtained by using ExEm-spFRET data d 、W a 、W s Then determining the W of cells transfected with different FRET plasmids alone s /W a And W s /W d Range R SA And R SD (ii) a Then, a template for data screening is manufactured according to the range and aiming at the imaging visual field, data screening is carried out, a straight line is fitted, and a correction factor K of the ExEm-spFRET system is obtained A /K D And Q A /Q D . The method can finish the measurement of the correction factor only by one cell sample, avoids the problem that different FRET plasmids have different contrasts and background signals when being measured independently, improves the accuracy of the correction parameters of the measurement system, reduces the use threshold and improves the efficiency.
Description
Technical Field
The invention belongs to the technical field of Fluorescence Resonance Energy Transfer (FRET) detection, and particularly relates to a method for measuring a correction factor of an excitation emission spectrum linear separation quantitative FRET system based on a cell sample of the same system and application thereof.
Background
FRET microscopy based on Fluorescent Proteins (FPs) has become an important tool for studying biochemical molecular dynamic processes in living cells. Obtaining a quantitative FRET signal independent of the detection system and the expression level of FPs is a necessary requirement for academic communication. Recently we developed a quantitative FRET measurement technique (ExEm-spFRET) based on simultaneous separation of excitation emission spectra, which can overcome both acceptor excitation crosstalk and donor emission crosstalk [ Mengyan Du, et al, "Wide-field microscopical FRET imaging using a single and multiple excitation spectroscopy", Opt. Express24(14), 16037-.
Accurate measurement of acceptor-donor quantum yield ratio (Q) A /Q D ) Ratio of the extinction coefficient of the acceptor-donor (K) A /K D ) Two parameters are key to quantitative ExEm-spFRET imaging. Most FRET-related studies were conducted by obtaining Q from the quantum yield values of fluorophores reported in the cited literature A /Q D The value is obtained. But in practice the Q of FRET measurement systems A /Q D The value is not only related to the optical properties of the donor and acceptor fluorescent groups, but also related to the intracellular environment of the fluorescent groups and the spectral response performance of the emission channel of the measurement system; k A /K D Related to the spectral properties of the excitation light source, the transfer function of the excitation channel of the measurement system, and the absorption spectra of the donor and acceptor. Especially for live cell quantitative ExEm-spFRET imaging, Q can be accurately measured A /Q D And K A /K D The value of (a) is a prerequisite for quantitative ExEm-spFRET imaging of viable cells. For a given FRET fluorophore pair and measurement System, Q A /Q D And K A /K D Is a constant and therefore once measured, there is no need to measure every time a quantitative FRET imaging analysis of live cells is performed.
In the prior art, Zhang [ Zhang C, Lin F, Du M, et al, "Simultaneous measurement of quaternary yield and absorption ratio between the receptor and the donor by linear approximation excitation spectrum", Journal of Microcopy 270(3), 335-; chen Tong Sheng, Zhang Chen Shuang, Lin Square Rui' a baseMethod of simultaneous measurement of acceptor-donor quantum yield ratio and extinction coefficient ratio in excitation emission spectroscopy separation "national invention patent 2019.7, issued patent no: ZL201710649949.8]A method for simultaneously measuring the quantum yield ratio and extinction coefficient ratio of donor and acceptor by transfecting cell samples expressing different FRET efficiencies and having the same donor concentration ratio with 4 dishes, respectively, is proposed. The method follows the excitation emission spectrum basis vector (S) of the donor D ) Excited emission spectrum basis (S) of the acceptor A ) And a basis vector (S) of excitation emission spectrum for receptor sensitization S ) The three spectrum basis vectors are subjected to linear separation to obtain three weight factors W d 、W a And W s . Then W a /W d As an independent variable, with W s /W d As dependent variable, linear fitting is carried out to obtain the inverse slope of the linear equation which is K A /K D The absolute value of the intercept is Q A /Q D . However, the method needs to switch a plurality of cell samples, is complex to operate, and cannot ensure the same contrast and background signal when four cell samples are imaged respectively, so that the method for measuring the correction factor of the ExEm-spFRET system in one cell sample is of great significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for measuring the linear separation and quantification of a FRET system correction factor based on the same system cell sample.
The invention also aims to provide application of the method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excitation emission spectrum of the cell sample in the same system.
The purpose of the invention is realized by the following technical scheme:
a method for measuring a correction factor of a linear separation quantitative FRET system (namely an ExEm-spFRET system correction factor) of an excitation emission spectrum based on a cell sample of the same system comprises the following steps:
(1) sample preparation
Firstly, a sample to be detected: 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);
reference sample: respectively transfecting cells with donor plasmids and acceptor plasmids, and obtaining a reference sample after the transfected plasmids are successfully expressed;
(2) ExEm-spFRET spectroscopic imaging and determination of W for FRET plasmid transfected cells s /W a And W s /W d Range of proportional values
Thirdly, respectively carrying out ExEm-spFRET spectral imaging on the reference samples obtained in the step II to obtain the emission spectrum basic vector S of the donor D Basal vector S of excited emission spectrum of receptor A And a basis vector S of the excitation emission spectrum for receptor sensitization S ；
Fourthly, performing ExEm-spFRET spectral imaging on the sample to be detected obtained in the step one, and selecting M cells for measurement: starting from the 1 st cell, data obtained from ExEm-spFRET spectroscopic imaging and three basis vectors S obtained from step c D 、S A And S S Performing spectral linear separation, and obtaining three weight factors W of each pixel point in the cell region d1 、W a1 And W s1 Then calculating their proportional values W respectively s1 /W a1 And W s1 /W d1 Performing single-peak Gaussian fitting, and taking the peak value to obtain W of the 1 st cell s1 /W a1 And W s1 /W d1 Ratio value SA -1 And SD -1 (ii) a Repeating the above steps to obtain W of M cells respectively s /W a And W s /W d Proportional value, denoted SA -1 、SA -2 ……SA -M ，SD -1 、SD -2 ……SD -M (ii) a Wherein M is more than or equal to 80 (and is an integer);
fifthly, mixing W of each cell s /W a And W s /W d The proportional values are arranged according to the size, and W is carried out at intervals of 0.01-0.1 s /W a And W s /W d Frequency distribution statistics (as statistical histograms) and i-peak Gaussian fitting (i.e., based on i FRET plasmidsGaussian fit) is performed, the W at the gaussian peak is obtained s /W a And W s /W d Value, denoted as P SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i ；
Is at P SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i Adding or subtracting 0.01-0.1 as W s /W a And W s /W d Scale value range, or selecting the half-height width (full width at half maximum, half-peak width) of the peak as W according to the result of Gaussian fitting s /W a And W s /W d The range of the ratio values, from small to large, is denoted as R SA-1 、R SA-2 ……R SA-i And R SD-1 、R SD-2 ……R SD-i ；
(3) Making data screening template drawings
Seventhly, randomly selecting one view field from the sample to be detected obtained in the step (1) to perform ExEm-spFRET spectral imaging, and obtaining three basis vectors S according to the step (c) D 、S A And S S Performing spectral linear separation to obtain the weight factor W of each pixel point under the visual field d ˊ、W a ' and W s ', is denoted as W d-1 ˊ、W d-2 ˊ……W d-n ˊ，W a-1 ˊ、W a-2 ˊ……W a-n And W s-1 ˊ、W s-2 ˊ……W s-n And calculating a ratio value W thereof s ˊ/W a And W s ˊ/W d ', denoted SA -1 ˊ、SA -2 ˊ……SA -n ˊ，SD -1 ˊ、SD -2 ˊ……SD -n '; wherein n is the total number of pixel points (integer) in the visual field;
w obtained by the step (c) s /W a And W s /W d Range of ratio value and ratio value W obtained in step (c) s ˊ/W a And W s ˊ/W d Preparing a template map for screening the visual field data:
template 1: if SA -1 At the ratio value R SA-1 In range and SD -1 At the ratio value R SD-1 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 Value of ratio R SA-1 In range and SD -2 Value of ratio R SD-1 In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n At the ratio value R SA-1 In range and SD -n At the ratio value R SD-1 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0;
template 2: if SA -1 At the ratio value R SA-2 In range and SD -1 At the ratio value R SD-2 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 Value of ratio R SA-2 In range and SD -2 At the ratio value R SD-2 In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n Value of ratio R SA-2 In range and SD -n At the ratio value R SD-2 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0;
……
template i: if SA -1 At the ratio value R SA-i In range and SD -1 At the ratio value R SD-i In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 At the ratio value R SA-i In range and SD -2 At the ratio value R SD-i In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n Value of ratio R SA-i In range and SD -n At the ratio value R SD-i In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0;
(4) obtaining correction factor of ExEm-spFRET system
Ninthly, according to the data screening result in the step (3), calculating as follows:
y 1-1 ＝W s-1 ˊ/W d-1 ' X template 1, x 1-1 ＝W a-1 ˊ/W d-1 ' template 1;
y 1-2 ＝W s-2 ˊ/W d-2 ' X template 1, x 1-2 ＝W a-2 ˊ/W d-2 ' template 1;
……
y 1-n ＝W s-n ˊ/W d-n ' X template 1, x 1-n ˊ＝W a-n ˊ/W d-n ' template 1;
calculating y 1-1 、y 1-2 ……y 1-n ，x 1-1 、x 1-2 ……x 1-n Average value of' noted as y 1 ' and x 1 ′；
By analogy, calculating to obtain an average value y 2 ′、y 3 ′……y i ' and x 2 ′、x 3 ′……x i ′；
Will y 1 ′、y 2 ′……y i ' (in ordinate) and x 1 ′、x 2 ′……x i ' (in abscissa) is fitted with a straight line by least square method, and the reciprocal and intercept of the slope of the fitted straight line are the ratio K of the extinction coefficients of the acceptor and the donor A /K D And acceptor-donor quantum yield ratio Q A /Q D ；
C, repeating the steps of R and R, optionally selecting N different fields of view from the sample to be detected obtained in the step (1) to carry out ExEm-spFRET spectral imaging, and obtaining Q from the N different fields of view A /Q D And K A /K D Taking an average value to obtain a correction factor K of the ExEm-spFRET system A /K D And Q A /Q D (ii) a Wherein N is more than or equal to 10.
The cell samples of the same system are cultured in the same container carrier; wherein, the container carrier can be a container carrier which can realize cell culture and imaging in the field, including but not limited to cell culture dish, well plate, slide glass, etc.; the invention selects the conventional cell culture dish to culture and image the cells.
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 step (1) is a container carrier capable of realizing cell culture and imaging, and includes, but is not limited to, a cell culture dish, a well plate, a glass slide and the like.
The donor plasmid in the step (1) is a donor plasmid cerulean (C); preferred is donor plasmid cerulean (c) purchased from the addgene plasmid pool, usa.
The acceptor plasmid in the step (1) is acceptor plasmid Venus (V); preferred is the recipient plasmid venus (v) available from the addgene plasmid pool, usa.
The cells in the first step (1) and the second step are preferably Hela cells.
The excitation emission spectrum basic vector (S) of the donor in the third step D ) Basal vector (S) of excited emission spectrum of acceptor A ) And a basis vector (S) of excitation emission spectrum for receptor sensitization S ) Is obtained by the following method: respectively carrying out ExEm-spFRET spectral imaging on the reference samples to obtain normalized donor excitation spectrum
And emission spectra
And normalizing the excitation spectrum of the receptor
And emission spectra
Then calculating the basic vector (S) of the excitation emission spectrum of the donor according to the formula (1) D ) Basal vector (S) of excited emission spectrum of acceptor A ) And a basis of stimulated emission Spectrum (S) for receptor sensitization S )。
The selection of the M cells in the step (2) is preferably realized by the following method: placing a sample container carrier (such as a culture dish) containing cells with transfected plasmids under a wide-field fluorescence microscope, selecting N' visual fields, selecting cells in each visual field, and selecting M cells for measurement; wherein N' is more than or equal to 40 (and is an integer); i.e., M cells are from N' fields of view (all cells in the carrier. gtoreq. all cells in N fields of view. gtoreq. M cells).
The value range of N' is preferably as follows: n' is more than or equal to 50.
The number of the selected cells in each field may be 1 to 3 (preferably 2 to 3).
The value range of M in the step (2) is as follows: m is more than or equal to 100; preferably: m is more than or equal to 500; since the cell proportion of the various transfected plasmids in the test sample is unknown, the larger the number of cells selected, the better.
The full width at half maximum of the peak value in the step (2) is preferably obtained by the following method:
p to be obtained SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i Counting at intervals of 0.01-0.1, making histogram, and recording the highest frequency as R SA And R SD Then R is added SA Or R2 SD Left and right R corresponding to the position/2 SA Or R SD The closed interval of the value interval is the full width at half maximum of the peak value.
The interval can be set according to actual conditions, and is preferably 0.03-0.05; wherein R is SA Preferably 0.03, R SD The interval of (3) is preferably 0.05.
The value range of N in the R in the step (4) is preferably as follows: n is more than or equal to 15.
The method for measuring the linear separation and quantification of the correction factor of the FRET system based on the excitation emission spectrum of the cell sample in the same system is applied to the technical field of Fluorescence Resonance Energy Transfer (FRET) detection.
The application is used for measuring the correction factor K of the ExEm-spFRET system A /K D (ratio of acceptor-donor extinction coefficients) and Q A /Q D (acceptor-donor quantum yield ratio).
The basic principle of the invention is as follows:
(1) obtaining an excitation emission basis vector by using a single-transfer donor sample and a single-transfer acceptor sample: excitation spectra by normalized donor
And emission spectra
And normalizing the excitation spectra of the receptors
And emission spectrum
The emission spectrum basis vector (S) of the donor can be obtained D ) Basal vector (S) of excited emission spectrum of acceptor A ) And a basis vector (S) of excitation emission spectrum for receptor sensitization S )：
(2) For a FRET sample, when the FRET sample contains both a free donor and a free acceptor, and a donor-acceptor in a FRET pair, the excitation emission spectrum of the FRET sample can be linearly divided into four excitation emission spectra: excitation emission spectrum of free donor, excitation emission spectrum of donor binding pair with acceptor, excitation emission spectrum for acceptor sensitization and excitation emission spectrum for direct excitation acceptor, as shown in formula (2):
wherein E is the FRET efficiency between the paired donor-acceptor in the FRET sample; c d Is the concentration of free donor in the FRET sample; c da Is the concentration of the donor-acceptor pair;
is the total concentration of acceptor in the FRET sample;
(3) simplifying equation (2) yields:
wherein, K A Is the receptor extinction coefficient; k D Is the donor extinction coefficient; q A Is the quantum yield of the acceptor; q D Is the quantum yield of the donor;
is the total concentration of donors in FRET
And has:
(4) apparent FRET efficiency (E) normalized by acceptor concentration A ) And apparent FRET efficiency normalized by donor concentration (E) D ) Can be expressed as:
(5) simultaneous equations (4) and (5) can be obtained:
(6) for the FRET tandem structure sample containing 1 donor and 1 acceptor (number ratio 1: 1), there is E A ＝E D Then equation (6) can be written as:
(7) multiple sets (W.sub.1) can be obtained by measuring 2 or more samples of FRET tandem structure containing 1 donor and 1 acceptor (number ratio 1: 1) A /W D ，W S /W D ) And through the pair W A /W D (abscissa) and W S /W D (ordinate) linear fitting is carried out, and the slope and intercept of the fitting straight line are respectively K in the formula (7) A /K D (ratio of acceptor-donor extinction coefficients) and Q A /Q D (acceptor-donor quantum yield ratio).
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 the ratio of acceptor-donor quantum yield (Q) A /Q D ) And the ratio of the extinction coefficients of the acceptor and donor (K) A /K D ) The method specifically comprises the following steps: obtaining a weight factor graph W by using ExEm-spFRET data d 、W a 、W s (ii) a Determination of W for cells transfected with different FRET plasmids alone s /W a And W s /W d Range R SA And R SD (ii) a Aiming at an imaging visual field, a template for data screening is manufactured; and screening data, fitting a straight line and obtaining a system correction factor.
(2) The invention provides a correction parameter Q of a simultaneous automatic measurement system based on automatic cell classification and data screening A /Q D And K A /K D Thereby improving the correction parameter Q of the measurement system A /Q D And K A /K D Is the development of intelligence and accuracyBasis for an intelligent FRET correction system.
(3) Q obtained by the invention A /Q D And K A /K D More accurate and is suitable for different detection systems, so the invention can greatly promote the application range of the ExEm-spFRET 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 (without the need of respectively and independently measuring each plasmid, and the problems of different contrasts and background signals existing in the respective and independent measurements).
Drawings
FIG. 1 shows the determination of different plasmids W according to the invention s /W a And W s /W d Process and result plots of ranges; wherein, a is ExEm-spFRET spectral imaging of a cell sample co-transformed with four plasmids; b is the weight ratio W obtained by spectral separation s /W a And W s /W d A pseudo-color map of the scale values; c and d are cell areas circled by red boxes in the selected pseudo-color image and are subjected to pixel-by-pixel W s /W a And W s /W d Counting the frequency distribution of the values and obtaining a fitting graph by single-peak Gaussian fitting; e and f are W pixel by pixel for a total of 589 cells in 92 fields, respectively s /W a And W s /W d And counting the frequency distribution, and carrying out four-peak Gaussian fitting to obtain a fitting graph.
FIG. 2 is a diagram of a system calibration parameter Q measured and obtained by the method of the present invention A /Q D And K A /K D The process and result diagram of (1); wherein, a is the data of ExEm-spFRET spectral imaging of any one visual field of four different plasmid cell samples transfected by one culture dish; b is according to R SA-1 、R SA-2 、R SA-3 、R SA-4 And R SD-1 、R SD-2 、R SD-3 、R SD-4 Data of range productionThe merge graph of the selected template (in the figure: red is data of cell transfection with CTV plasmid, blue is data of cell transfection with C17V plasmid, green is data of cell transfection with C32V plasmid, and purple is data of cell transfection with C5V plasmid); c and d are respectively W of the visual field data after screening s /W d And W s /W a A pseudo-color image; e is a straight line graph (Q) obtained by fitting four plasmids according to a least square method A /Q D 2.0676 and K A /K D ＝0.1619)。
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. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as 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 formed by linking C and V through a 32-amino acid connecting sequence;
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: donor plasmids cerulean (C), acceptor plasmids venus (v), and FRET reference plasmids C5V, C17V, C32V, 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 containing 5% (v/v) carbon dioxide at 37 ℃. Digesting the cells by trypsin, transferring the cells to a cell culture dish, culturing the cells for 24 hours, and then using an in vitro transfection reagent Turbofect when the cells grow to 70-90 percent TM The 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 culture medium into the EP tube, then adding 1-2 mu L of transfection reagent into the EP tube, then adding 1-2 mu L (500-600 ng/mu L) of plasmid into the EP tube, 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) respectively carrying out ExEm-spFRET imaging after completing the steps (1) to (3) on samples of a single-transfer donor plasmid Cerulean (C) and a single-transfer acceptor plasmid Venus (V) to obtain fingerprint spectrums of the donor and the acceptor, namely, basic vectors of the excitation emission spectrum of the donor (S) D ) Basal vector (S) of excited emission spectrum of acceptor A ) And a basis vector (S) of excitation emission spectrum for receptor sensitization S )。
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 ExEm-spFRET spectroscopic imaging. The wide field fluorescence microscope was configured as follows: light of 436/20nm and light of 470/20nm are respectively used as exciting light of Cerulean and Venus, and channels of 470/20nm, 490/20nm, 510/20nm, 530/20nm and 550/20nm are respectively used as detection channels of Cerulean and Venus fluorescence. Under the excitation of 436/20nm, the detector collects the images of all detection channels, under the excitation of 470/20nm, the detector only collects the images of 510/20nm, 530/20nm and 550/20nm detection channels, and a total of 8 images are recorded as ExEm-spFRET data of one field of view.
5. Data processing
5.1 ExEm-spFRET data from multi-plasmid cotransferred samples and the excitation emission spectra basis (Donor excitation emission Spectrum (S) D ) Acceptor excitation emission spectrum (S) A ) And acceptor-sensitized excitation emission spectrum (S) S ) Linear separation to obtain the weight factor graph W of each component d 、W a And W s The specific process is as follows:
in the step of4 multiple plasmid cotransformation sample (same culture dish) selecting 92 fields at random, selecting cells under each field, selecting 589 cells for measurement (the larger the number of cells selected, the better because the ratio of cells of various transfection plasmids in the multiple plasmid cotransformation sample is unknown), obtaining the emission spectra of the sample under the excitation of 436/20nm and 470/20nm, and obtaining the spectra S of the donor and the acceptor according to the above steps 3 and 4) D 、S A And S S (i.e. using single-transfer donor plasmid cerulean (C) and single-transfer acceptor plasmid Venus (V) as reference sample), and the spectrum base vector obtained by calculation of formula (1), making linear separation of spectrum according to formula (3), respectively calculating to obtain weight factor W d 、W a And W s ；
Here, the measurement can be carried out under the same dish, i.e., after the Hela cells transfected with CTV, C5V, C17V and C32V alone are mixed into the same dish; the measurement of the individually transfected CTV, C5V, C17V and C32V Hela cells (different culture dishes) may be performed on individually selected cells (the number of selected cells may be selected as the case may be, preferably, 10 or more fields (preferably 20 or more) are selected for each culture dish transfected with different plasmids, and 1 or more cells (preferably 3 to 7 cells) are selected for each field), and the single transfer donor plasmid Cerulean (C) and the single transfer recipient plasmid Venus (V) are used as reference samples, based on the measured S D 、S A And S S And spectrum basis vectors obtained by calculation of the formula (1) are subjected to linear separation of spectra according to the formula (3), and weight factors W are obtained by calculation respectively d 、W a And W s (the respective measurements here are such that there is a different background signal subtraction, but the effect on the results is not significant). This experiment selects for measurement selected cells in the same dish, i.e. directly in the multi-plasmid cotransformation sample in step 4.
5.2 determination of W for 4 FRET plasmid transfected cells s /W a And W s /W d Proportional value (R) SA And R SD ) The range is as follows:
5.2.1 Single Peak Gaussian fitting:
for a total of 589 cells in 92 fields, W was performed pixel by pixel for each cell s /W a And W s /W d Counting frequency distribution, and carrying out single-peak Gaussian fitting to obtain W at a Gaussian peak s /W a And W s /W d The values are specifically:
as shown in FIG. 1a, after the ExEm-spFRET spectral imaging is performed on the cell sample co-transformed with four plasmids, the ExEm-spFRET data of the sample is subjected to spectral separation to obtain the weight ratio W s /W a And W s /W d A graph of the proportional values (fig. 1 b); the cell area circled by the red box in FIG. 1b is then selected and subjected to a pixel-by-pixel W s /W a And W s /W d The frequency distribution of the values is counted (namely in the cell region, from the 1 st image point, the ExEm-spFRET data is obtained, and the weight factor W of each pixel is obtained according to the emission spectrum and the basis vector of the sample under the excitation of 436/20nm and 470/20nm s 、W a And W d Then calculate W s /W a And W s /W d A value; and so on until the last pixel point of the cell region (the size of the map used in the present invention is 2048 × 2048 pixels (pixels)); wherein the weight factor W s 、W a And W d The method of obtaining (1) is the same as that of (5.1), using single transfer donor plasmid Cerulean (C) and single transfer acceptor plasmid Venus (V) as reference samples, and performing single-peak Gaussian fitting (FIGS. 1c and 1d) to obtain W of the cell s /W a And W s /W d Value, i.e. SA -1 1.6443 and SD -1 0.94024 (statistical blue points in fig. 1c and 1d were gaussian fitted using matlab software to obtain a gaussian function (red line) with the peak of the gaussian function).
The same procedure as described above was used to obtain a weighting factor W for 589 cells d 、W a And W s And calculating the SA and SD values of each cell (SA ═ W) s /W a And SD ═ W s /W d ) Are respectively denoted as SA -1 、SA -2 ……SA -589 ，SD -1 、SD -2 ……SD -589 。
5.2.2 four-peak Gaussian fitting:
SA of each cell -1 、SA -2 ……SA -589 ，SD -1 、SD -2 ……SD -589 Respectively arranging according to the numerical value from small to large, and then carrying out W of different cells at intervals of 0.01-0.1 (in the experiment, the interval is 0.03 for SA, and the interval is 0.05 for SD) s /W a And W s /W d And counting the frequency distribution, and performing four-peak Gaussian fitting. As shown in FIGS. 1e and 1f, we judged the relative strengths of the efficiencies of the different FRET plasmids (C5V, C17V, C32V, CTV) (the higher the FRET efficiency, the higher the ratio R SA And R SD The larger), the red gaussian peak fitted to the figure was the cell transfected with CTV plasmid, the blue gaussian peak fitted to the cell transfected with C32V plasmid, the green gaussian peak fitted to the cell transfected with C17V plasmid, and the purple gaussian peak fitted to the cell transfected with C5V plasmid.
Here, it should be noted that: in the same culture dish, for the FRET standard plasmid with known FRET efficiency or known FRET efficiency relative to strength, the higher the FRET efficiency is, the ratio value R of the FRET standard plasmid is SA And R SD The larger (according to equation (6): standard plasmid with donor to acceptor ratio 1: 1, ED ═ EA, EA proportional to W S /W A Similarly, ED is proportional to W S /W D Therefore, the higher the FRET efficiency, the larger the ratio), i.e., the FRET plasmids with 4 different efficiencies as mentioned above, the FRET efficiency is: FRET C5V ＞FRET C17V ＞FRET C32V ＞FRET CTV The proportion value is as follows: r SA-C5V ＞R SA-C17V ＞R SA-C32V ＞R SA-CTV ，R SD-C5V ＞R SD-C17V ＞R SD-C32V ＞R SD-CTV Thus, it can be determined in FIG. 1 which FRET plasmid corresponds to the Gaussian peak. ② in the same culture dish, for the FRET standard plasmid with unknown FRET efficiency, only need to obtain its peak value according to Gaussian fitting, as shown in figure 1, can distinguish four FRET plasmids.
The method carries out Gaussian fitting twice, can ensure that the data is more accurate, carries out pixel-by-pixel statistics on the cells for the first time, obtains peak values SA and SD by unimodal Gaussian fitting, and reduces the dimension of the data of one cell; carrying out statistics on SA and SD values of different cells for the second time, and then carrying out four-peak Gaussian fitting, so that the determined range is less influenced by unstable factors and is more accurate; on the other hand, if all cells are counted pixel by pixel directly and then all data are fitted together by the four-peak gaussian fitting, the characteristic peak may be buried and the fitting may not be accurate.
5.2.3 according to the results of FIG. 1, the peak P of 4 cells (cells transfected with CTV, C5V, C17V and C32V) was obtained SA And P SD In the experiment, the data corresponding to the peak values are respectively as follows: p is SA-1 ≈0.15、P SA-2 ≈1.40、P SA-3 ≈1.90、P SA-4 2.30 and P SD-1 ≈0.1、P SD-2 ≈0.6、P SD-3 ≈0.9、P SD-4 1.3, on the basis of this, i.e. at peak value P SA And P SD The ratio range R is defined as the range R of the ratio of 0.01 to 0.1 (preferably 0.05 to 0.1) at both ends of (A), and thus the range R of 4 plasmids can be determined SA-1 ∈(0.1,0.2)、R SA-2 ∈(1.35,1.55)、R SA-3 ∈(1.8,2.0)、R SA-4 E (2.2,2.4) and R SD-1 ∈(0.05,0.15)、R SD-2 ∈(0.5,0.7)、R SD-3 ∈(0.8,1.0)、R SD-4 E (1.2, 1.4). It should be noted that: at peak value P SA And P SD The two ends of (1) are added or subtracted by 0.01-0.1, and can be set according to actual conditions and according to W S /W A And W S /W D Under the two conditions, the range of the ratio of each FRET plasmid (the value of the peak value. + -. range) is as large as possible, but the mutual influence of the ranges of the ratio of each FRET plasmid is ensured to be minimum (the values of the peak values. + -. ranges of the four plasmids are ensured not to intersect). The above 4 FRET plasmids were synthesized in P based on the above results and FIG. 1 SA-1 、P SA-2 、P SD-1 Addition or subtraction of 0.05 on the basis of P SA-3 、P SA-4 、P SD-2 、P SD-3 、P SD-4 Adding or subtracting 0.1 on the basis of (1), and determining the proportional value range R of 4 plasmids SA And R SD 。
5.3 one field of the above-mentioned CTV, C5V, C17V and C32V co-transferred sample (arbitraryOne field of view is selected), the ExEm-spFRET spectral imaging data is shown in figure 2a, and the weight factor W of each pixel is obtained according to the emission spectrum and basis vector of the sample under the excitation of 436/20nm and 470/20nm s ˊ、W a And W d ' (weight factor W) s ˊ、W a And W d The acquisition method of' is the same as 5.1, and the single transfer donor plasmid cerulean (c) and the single transfer acceptor plasmid venus (v) are used as reference samples; is marked as W s-1 ˊ、W s-2 ˊ……W s-n ˊ；W a-1 ˊ、W a-2 ˊ……W a-n ˊ；W d-1 ˊ、W d-2 ˊ……W d-n '; where n is the total number of all pixels in the field of view, the size of the graph used in the present invention is 2048 × 2048 pixels (pixels)), using R obtained in 5.2 SA And R SD Template map for screening of the visual field data was generated (FIG. 2 b):
template 1: according to the obtained weight factor W of each pixel point s ˊ、W a And W d Get W pixel by pixel ″ s ˊ/W a In the range (0.1,0.2) (i.e. in the above range R) SA-1 ) In and W s ˊ/W d At range (0.05,0.15) (i.e., in the above range R) SD-1 ) If the conditions are met, setting the pixel point to be 1 (namely the value of the pixel point at the corresponding position in the template 1 is '1'), and otherwise, setting the pixel point to be 0 (namely the value of the pixel point at the corresponding position in the template 1 is '0'); that is, in the selected view, starting from the 1 st pixel point, if the proportion value R of the pixel point SA ˊ(R SA ˊ＝W s-1 ˊ/W a-1 ') is in the range (0.1,0.2) and R SD ˊ(R SD ˊ＝W s-1 ˊ/W d-1 ') is in the range (0.05,0.15), then the pixel point is set to 1, otherwise to 0; and so on until the last image number point under the view;
template 2: taking W pixel by pixel in the same manner as described above s ˊ/W a In the range (1.35,1.55) and W s ˊ/W d In the range (0.5,0.7), if the condition is satisfied at the same time, the pixel point is set to 1, otherwise, the pixel point is set to 0;
template 3: taking W pixel by pixel in the same manner as described above s ˊ/W a In the range (1.8,2.0) and W s ˊ/W d In the range (0.8, 1.0'), if the condition is satisfied at the same time, the pixel point is set to 1, otherwise, to 0;
and (4) template: taking W pixel by pixel in the same manner as described above s ˊ/W a Is in the range (2.2,2.4) and W s ˊ/W d In the range (1.2, 1.4'), if the condition is satisfied at the same time, the pixel point is set to 1, otherwise, it is set to 0.
FIG. 2b is a merge image of template 1, template 2, template 3, template 4; wherein, the red pixel points represent the data position of the CTV transfected by the screening cells of the template 1; blue pixel points represent data positions of template 2 selected cells transfected with C32V; the green pixel represents the data position of template 3 selected cell transfection C17V; purple dots indicate the position of data for C5V transfection of mod 4 selected cells. FIG. 2c and FIG. 2d are W after data screening, respectively s /W d And W s /W a A pseudo-color image of (2).
Note: in addition to data screening for the CTV, C5V, C17V, and C32V co-transformed samples, data screening was also performed for two or more of these 4 co-transformed samples, as described above.
5.4 the field data was screened, averaged and fitted to a straight line using 4 templates:
5.4.1 obtaining data of template 1, template 2, template 3 and template 4 according to 5.3 and formula (6), respectively calculating to obtain y 1 、y 2 、y 3 、y 4 And x 1 、x 2 、x 3 、x 4 Values, then averaging respectively; namely:
①y 1-1 ＝W s-1 ˊ/W d-1 ' X template 1, x 1-1 ＝W a-1 ˊ/W d-1 ' template 1;
y 1-2 ＝W s-2 ˊ/W d-2 ' X template 1, x 1-2 ＝W a-2 ˊ/W d-2 ' template 1;
……
y 1-n ＝W s-n ˊ/W d-n ' X template 1, x 1-n ˊ＝W a-n ˊ/W d-n ' template 1;
calculating y 1 (y 1-1 、y 1-2 ……y 1-n ) And x 1 (x 1-1 、x 1-2 ……x 1-n 'average value of') noted y 1 ' and x 1 ′；
② according to the same method, calculating to obtain average value y 2 ' and x 2 ′：
y 2-1 ＝W s-1 ˊ/W d-1 ' X template 2, x 1-1 ＝W a-1 ˊ/W d-1 ' template 2;
y 2-2 ＝W s-2 ˊ/W d-2 ' X template 2, x 1-2 ＝W a-2 ˊ/W d-2 ' template 2;
……
y 2-n ＝W s-n ˊ/W d-n ' X template 2, x 1-n ˊ＝W a-n ˊ/W d-n ' template 2;
calculating y 2 (y 2-1 、y 2-2 ……y 2-n ) And x 2 (x 2-1 、x 2-2 ……x 2-n 'average value of') noted y 2 ' and x 2 ′；
Thirdly, analogizing in sequence, and calculating to obtain an average value y 3 ′、y 4 ' and x 3 ′、x 4 ′。
The average values obtained in this experiment are as follows:
y 1 taking the average to obtain y 1 ′＝0.069，x 1 Taking the average to obtain x 1 ′＝0.359；
y 2 Taking the average to obtain y 2 ′＝0.607，x 2 Taking the average to obtain x 2 ′＝0.418；
y 3 Taking the average to obtain y 3 ′＝0.902，x 3 Taking the average to obtain x 3 ′＝0.474；
y 4 Taking the average to obtain y 4 ′＝1.266，x 4 Taking the average to obtain x 4 ′＝0.548；
Four sets of averaged data points (y) were obtained 1 ′，x 1 ′)、(y 2 ′，x 2 ′)、(y 3 ′，x 3 ′)、(y 4 ′，x 4 '), i.e. (0.359,0.069), (0.418,0.607), (0.474,0.902), (0.548, 1.266). By making a pair of W a /W d (abscissa) and W s /W d (ordinate) according to the formula
A least squares linear fit (FIG. 2e) is performed, with the slope and intercept of the fitted line being K in equation (6), respectively A /K D 0.1619 and Q A /Q D ＝2.0676。
5.4.2 the above experiment is a linear fit to the average values in any field, and finally the correction factor for this field is obtained. In order to obtain more accurate data of the correction factor, multiple measurements can be taken and averaged, for example, multiple fields (at least 10-15 fields) are selected, and Q is obtained in different fields according to the methods of 5.3 and 5.4 A /Q D And K A /K D Then, the mean value is taken to obtain the correction factor K of the ExEm-spFRET system A /K D And Q A /Q D (ii) a Also, the W of 589 cells obtained in 5.2.1 above can be used directly s /W a And W s /W d Values, corresponding to Q, obtained in the methods of 5.3 and 5.4 A /Q D And K A /K D Then, the mean value is taken to obtain the correction factor K of the ExEm-spFRET system A /K D And Q A /Q D 。
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, such as defined by the definition of W in the specification s /W a And W s /W d Value to classify cells, enter dataThe screening is performed, and in practical application, any method using the change of the spectral information to distinguish different transfected plasmids and the screening of data is included in the protection scope of the present invention.
Claims (10)
1. A method for measuring a calibration factor of an excited emission spectrum linear separation quantitative FRET system based on a cell sample of the same system is characterized by comprising the following steps:
(1) sample preparation
Firstly, a sample to be detected: 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 referring to a sample: respectively transfecting cells with donor plasmids and acceptor plasmids, and obtaining a reference sample after the transfected plasmids are successfully expressed;
(2) ExEm-spFRET spectroscopic imaging and determination of W for FRET plasmid transfected cells s /W a And W s /W d Range of proportional values
Thirdly, respectively carrying out ExEm-spFRET spectral imaging on the reference samples obtained in the step II to obtain the emission spectrum basic vector S of the donor D Basal vector S of excited emission spectrum of receptor A And a basis vector S of the excitation emission spectrum for receptor sensitization S ；
Fourthly, performing ExEm-spFRET spectral imaging on the sample to be detected obtained in the step one, and selecting M cells for measurement: starting from the 1 st cell, data obtained from ExEm-spFRET spectroscopic imaging and three basis vectors S obtained from step c D 、S A And S S Performing spectral linear separation, and obtaining three weight factors W of each pixel point in the cell region d1 、W a1 And W s1 Then calculating their proportional values W respectively s1 /W a1 And W s1 /W d1 Performing single-peak Gaussian fitting, and taking the peak value to obtain W of the 1 st cell s1 /W a1 And W s1 /W d1 Ratio value SA -1 And SD -1 (ii) a Is repeated onThe steps of obtaining W of M cells, respectively s /W a And W s /W d Proportional value, denoted SA -1 、SA -2 ……SA -M ，SD -1 、SD -2 ……SD -M (ii) a Wherein M is more than or equal to 80;
fifthly, mixing W of each cell s /W a And W s /W d The proportional values are arranged according to the size, and W is carried out at intervals of 0.01-0.1 s /W a And W s /W d Frequency distribution statistics and i peak Gaussian fitting to obtain W at the Gaussian peak s /W a And W s /W d Value, denoted as P SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i ；
Is at P SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i Adding or subtracting 0.01-0.1 as W s /W a And W s /W d Scale value range, or selecting the half-height width of the peak as W according to the result of Gaussian fitting s /W a And W s /W d The range of the proportional value is marked as R from small to large SA-1 、R SA-2 ……R SA-i And R SD-1 、R SD-2 ……R SD-i ；
(3) Making data screening template drawings
Seventhly, randomly selecting one view field from the sample to be detected obtained in the step (1) to perform ExEm-spFRET spectral imaging, and obtaining three basis vectors S according to the step (c) D 、S A And S S Performing spectral linear separation to obtain the weight factor W of each pixel point under the visual field d ˊ、W a And W s ', mark W d-1 ˊ、W d-2 ˊ……W d-n ˊ，W a-1 ˊ、W a-2 ˊ……W a-n And W s-1 ˊ、W s-2 ˊ……W s-n And calculating a ratio value W thereof s ˊ/W a And W s ˊ/W d ', denoted SA -1 ˊ、SA -2 ˊ……SA -n ˊ，SD -1 ˊ、SD -2 ˊ……SD -n '; wherein n is the total number of pixel points in the visual field;
w obtained by the step (c) s /W a And W s /W d Range of ratio value and ratio value W obtained in step (c) s ˊ/W a And W s ˊ/W d Preparing a template map for screening the visual field data:
template 1: if SA -1 At the ratio value R SA-1 In range and SD -1 At the ratio value R SD-1 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 At the ratio value R SA-1 In range and SD -2 At the ratio value R SD-1 In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n At the ratio value R SA-1 In range and SD -n At the ratio value R SD-1 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0;
template 2: if SA -1 At the ratio value R SA-2 In range and SD -1 At the ratio value R SD-2 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 At the ratio value R SA-2 In range and SD -2 At the ratio value R SD-2 In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n At the ratio value R SA-2 In range and SD -n At the ratio value R SD-2 In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0;
……
template i: if SA -1 At the ratio value R SA-i In range and SD -1 Value of ratio R SD-i In the range, the pixel point is set to be 1, otherwise, the pixel point is set to be 0; if SA -2 Value of ratio R SA-i In range and SD -2 At the ratio value R SD-i In the range, the pixel point is set to 1, otherwise, the pixel point is set to 0 … …, and so on, if SA -n At the ratio value R SA-i In range and SD -n At the ratio value R SD-i In the range, the pixel point is setIs 1, otherwise is set to 0;
(4) obtaining correction factor of ExEm-spFRET system
Ninthly, according to the data screening result in the step (3), calculating as follows:
y 1-1 ＝W s-1 ˊ/W d-1 ' X template 1, x 1-1 ＝W a-1 ˊ/W d-1 ' template 1;
y 1-2 ＝W s-2 ˊ/W d-2 ' X template 1, x 1-2 ＝W a-2 ˊ/W d-2 ' template 1;
……
y 1-n ＝W s-n ˊ/W d-n ' X template 1, x 1-n ˊ＝W a-n ˊ/W d-n ' template 1;
calculating y 1-1 、y 1-2 ……y 1-n ，x 1-1 、x 1-2 ……x 1-n Average value of' noted as y 1 ' and x 1 ′；
By analogy, calculating to obtain an average value y 2 ′、y 3 ′……y i ' and x 2 ′、x 3 ′……x i ′；
Will y 1 ′、y 2 ′……y i ' and x 1 ′、x 2 ′……x i Fitting a straight line by a least square method, wherein the reciprocal and the intercept of the slope of the fitted straight line are the ratio K of the extinction coefficients of the receptor and the donor A /K D And acceptor-donor quantum yield ratio Q A /Q D ；
C, repeating the steps of R and R, optionally selecting N different fields of view from the sample to be detected obtained in the step (1) to carry out ExEm-spFRET spectral imaging, and obtaining Q from the N different fields of view A /Q D And K A /K D Taking an average value to obtain a correction factor K of the ExEm-spFRET system A /K D And Q A /Q D (ii) a Wherein N is more than or equal to 10.
2. The method for measuring the calibration factor of the quantitative FRET system based on the linear separation of the excited emission spectra of the cell samples in the same system according to claim 1, wherein:
the types of i in the step (1) are 2-4;
the value range of M in the step (2) is as follows: m is more than or equal to 100;
the value range of N in the step (4) R is as follows: n is more than or equal to 15.
3. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 2, wherein:
the types of i in the step (1) are 4;
the value range of M in the step (2) is as follows: m is more than or equal to 500.
4. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 1, which is characterized in that:
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 donor plasmid in the step (1) is donor plasmid Cerulean;
the acceptor plasmid in the step (1) is acceptor plasmid Venus.
5. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 4, wherein:
the number ratio of the FRET plasmid as a donor to an acceptor in the (1) part is 1: 1, in a cell culture medium.
6. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 5, wherein:
the FRET plasmid in the step (1) is at least two of C5V, C17V, C32V and CTV.
7. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 1, which is characterized in that:
the selection of the M cells in the step (2) is realized by the following mode: placing the sample container carrier containing the cells with the transfection plasmids under a wide-field fluorescence microscope, selecting N' visual fields, selecting cells in each visual field, and selecting M cells for measurement; wherein N' is more than or equal to 40 and is an integer;
the half-height width of the peak value in the step (2) is obtained by the following method: p to be obtained SA-1 、P SA-2 ……P SA-i And P SD-1 、P SD-2 ……P SD-i Counting at intervals of 0.01-0.1, making a histogram, and recording the highest frequency as R SA And R SD Then R is added SA Or R2 SD Left and right R corresponding to the position/2 SA Or R SD The closed interval of the value interval is the full width at half maximum of the peak value.
8. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excited emission spectrum of the cell sample in the same system according to claim 1, which is characterized in that:
the cells in the first step and the second step are Hela cells;
the sample container carrier in the step (1) is one of a cell culture dish, a pore plate and a glass slide.
9. The method for measuring the correction factor of the quantitative FRET system based on the linear separation of the excitation emission spectrum of the cell sample of the same system as in any one of claims 1 to 8 is applied to the technical field of fluorescence resonance energy transfer detection.
10. Use according to claim 9, characterized in that:
the application is used for measuring the correction factor K of an ExEm-spFRET system A /K D And Q A /Q D 。
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