CN113899722B - Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof - Google Patents
Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof Download PDFInfo
- Publication number
- CN113899722B CN113899722B CN202111083669.8A CN202111083669A CN113899722B CN 113899722 B CN113899722 B CN 113899722B CN 202111083669 A CN202111083669 A CN 202111083669A CN 113899722 B CN113899722 B CN 113899722B
- Authority
- CN
- China
- Prior art keywords
- fret
- plasmid
- beta
- donor
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a method for measuring a FRET system correction parameter based on a single standard FRET plasmid and application thereof. The method only needs to carry out three-channel fluorescence imaging on the cells transfected with one standard FRET plasmid, and can calculate and obtain the correction parameters beta, lambda, G factor and k factor of the FRET system through physical analysis and a mathematical calculation equation, so that at least four experimental samples which are originally needed to be used are reduced to one, the number of biological samples which are needed to be used is greatly reduced, the experimental efficiency is improved, and the method has high convenience, stability and reliability.
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 FRET system correction parameter based on a single standard FRET plasmid and application thereof.
Background
Quantitative FRET microscopy has become an important tool for studying the dynamic processes of biochemical molecules in living cells. The quantitative FRET detection technology can be used for real-time dynamic research of molecule combination and separation in living cells, and can also be used for measuring the information ratio of biological single molecule structures in the living cells.
FRET microscopy based on a combination of three filters (3-cube FRET microscopy, E-FRET method for short). Three images of the FRET sample are required to be obtained using three different sets of fluorescence filters, respectively: donor channel image (I)DDDonor fluorescence detected in the donor fluorescence detection channel upon excitation with donor excitation light), acceptor channel image (I)AAAcceptor fluorescence detected in the acceptor fluorescence detection channel upon excitation with acceptor excitation light), FRET channel image (I)DAFluorescence detected in the acceptor fluorescence detection channel upon excitation by donor excitation light).
The systematic correction parameters (a, b, c, d, G-factor and k-factor) are key parameters for quantitative FRET detection. a. b, c and d represent fluorescence crosstalk spectral coefficients between donor and acceptor spectra; g factor indicates the fluorescence emitted by the sensitized acceptor (F)c) The ratio of fluorescence to fluorescence at which the donor is quenched due to FRET; the k factor represents the ratio of the fluorescence intensity of the donor acceptor at the same molar concentration in the absence of FRET. For a given FRET fluorophore pairLike the system, the system correction parameter is constant. Measurement to obtain reliable systematic correction parameters a, b, c, d, G-factor and k-factor is key to quantitative FRET measurement.
The current method is mainly to measure four spectral crosstalk coefficients a, b, c, d by separately transfecting donor and acceptor cells respectively, and to measure factor G and factor k using standard FRET plasmids. Researchers have developed a variety of methods for measuring factor G. Zal and Gascoigne [ T.Zal and N.R.J.Gascoigne, "Photostabilized-corrected FRET interference of live cells," biophys.J.86 (6), 3923-3939 (2004) ] propose a method for determining G-factor based on gradual bleaching of receptors that immobilize FRET cell samples, which requires simultaneous measurement of images of three channels of FRET samples before and after bleaching, at least 4 times by switching filter sets (cube). Nagy et al [ Nagy, peter, et al, "Novel catalysis method for flow Cytometry luminescence reactions between visible luminescence proteins," cytometric Part a67 (2), 86-96 (2005) ] determine G-value by three FRET tandem structures with donor-acceptor concentration ratio of 1 and different E-values, which requires many samples of living cells, the experimental process is cumbersome, and the data processing is difficult. Chen et al [ H.Chen et al, "Measurements of FRET efficacy and Ratio of Donor to Acceptor control in living Cells," Biophys.J.91 (5), L39-L41 (2006) ] propose the determination of factor G in living Cells using two FRET tandem structures with an Acceptor Concentration Ratio of 1 and an unknown E value, which requires the preparation of two FRET living cell samples of different efficiencies, and both FRET samples must be measured under identical conditions. The subject group [ J.Zhang et al "," replaceable measurement of the FRET sensitive-quantification in the cells "," Micron 88,7-15 (2016) ] has proposed a method for measuring the G value of two cells transfected with different plasmids in the same cell culture dish, ensuring that the measurement of cells expressing two FRET plasmids is performed under the same conditions, realizing data acquisition and calculation of the G value of one dish of cells, and improving the experimental efficiency to a certain extent. Although researchers have improved the measurement methods to obtain accurate system correction parameters, the actual measurement process still requires that cells of donor and recipient are transfected separately and two or more plasmids are transfected, and at least four experimental samples are used, which is complicated and needs to be improved in efficiency. This is detrimental to the performance of FRET measurements in living cells, preventing the wider use of FRET techniques.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for measuring a FRET system correction parameter based on a single standard FRET plasmid.
Another object of the present invention is to provide an application of the method for measuring the correction parameter of the FRET system based on the single standard FRET plasmid.
The purpose of the invention is realized by the following technical scheme:
a method for measuring a FRET system correction parameter based on a single standard FRET plasmid comprises the following steps:
(1) Obtaining three-channel average fluorescence intensity data by circling cell area
Knowing the efficiency value E1Concentration ratio to donor acceptor R1After the transfected plasmids are successfully expressed, n cells are selected to respectively carry out three-channel fluorescence imaging to obtain three-channel average fluorescence intensity IDD、IAA、IDAImages, then at IDD、IAA、IDASelecting m different cell areas in the cell inner circle in the image, and respectively obtaining three channels of average fluorescence intensity IDD1、IAA1、IDA1，IDD2、IAA2、IDA2......IDDm、IAAm、IDAmAnd the mean fluorescence intensity I of three channels obtained from the 1 st cell regionDD1、IAA1、IDA1Three-channel mean fluorescence intensity I obtained for the 2 nd cell region for the first subset of dataDD2、IAA2、IDA2Three-channel mean fluorescence intensity I obtained for the m-th cell region for the second subset of data, and so onDDm、IAAm、IDAmFor the m-th sub-group of data,taking the data of 2 cell areas as a group, and dividing the data into t groups in pairs; wherein n is a positive integer and n is more than or equal to 1, m is more than or equal to 2, t is more than or equal to 1;
(2) Calculating FRET system correction parameters beta, lambda, G factor and k factor
(1) Calculating a FRET system correction parameter beta according to a first group of data in the t groups of data obtained in the step (1) and the following equation1，λ1，G1And k1：
minf(β1，λ1，G1，k1)＝|y1|+|y2|+|y3|+|y4|；
β1＞0；λ1＞0；G1＞0；k1＞0；
λ′+e＞λ1＞λ′-e；
β′+e1＞β1＞β′-e1；
0＜e1＜1；0＜e＜1；
Wherein:
λ' is the maximum efficiency value (e) at the intersection of the donor emission spectrum and the acceptor emission filter spectral passbandDa) Maximum efficiency value (e) at intersection with donor emission spectrum and donor emission filter spectral passbandDd) Ratio (λ' = e) ofDa/eDd)；
Beta' is the highest efficiency value (e) at the intersection of the acceptor excitation spectrum and the spectral passband of the donor excitation filterdA) Maximum efficiency value (e) at intersection with the receptor excitation spectrum and the spectral passband of the receptor excitation filteraA) Multiplied by the resulting value of the relative intensity ratio phi of the illumination of the donor excitation and the acceptor excitation (beta' = (e)dA/eaA)*φ)；
(2) Replacing the first group of data in the step (1) with the second group of data, and calculating the FRET system correction parameter beta according to the equation2，λ2，G2And k2(ii) a And analogizing until the FRET system correction parameter beta of the t-th group of data is obtained by calculationt，λt，GtAnd kt(ii) a Finally, respectively calculating beta1、β2......βt，λ1、λ2......λt，G1、G2......GtAnd k1、k2......ktAverage value of (beta)v，λv，Gv，kv(ii) a Beta is beta ofv，λv，Gv，kvNamely, the FRET system correction parameters beta, lambda, G factor and k factor.
The standard FRET plasmid in the step (1) can be directly purchased with a known efficiency value and donor-acceptor concentration ratio; wherein, the efficiency value can be measured in advance by a life measuring method, an E-FRRT measuring method or a spectral linear separation method and the like; the number (concentration) ratio of the donor to the acceptor is 1: N, and N is more than or equal to 1 (the number (concentration) ratio of the donor to the acceptor is preferably 1: 1); further preferred is any of standard FRET plasmids C5V, C17V, C32V and CTV (FRET tandem plasmid constructs consisting of a 229 amino acid linker linking C and V, commercially available from the Addge plasmid library, USA).
Efficiency E of C32V1＝0.31。
The efficiency value E of C5V2＝0.46。
The efficiency value E of C17VD＝0.38±0.03。
The cells described in step (1) are preferably Hela cells.
The cell region described in step (1) has no special requirement, and can be freely selected and the size of the selected cell region can be controlled, for example, the cells in one image are traversed from top to bottom and from left to right by a program, one cell region (r is preferably 10) is determined by r pixels, and then the cell region is randomly selected from all the cell regions, namely the cell region is in the cell, can contain the whole cell, and is also a part of the cell.
The three-channel imaging in the step (1) is to acquire a three-channel image by using a FRET microscopic imaging system based on the combination of three optical filters.
The three-channel average fluorescence intensity in the step (1) is the three-channel average fluorescence intensity after background subtraction, and specifically comprises the following steps: the cell-free area was taken as background and background subtraction was performed.
The value range of n in the step (1) is preferably that n is more than or equal to 10; more preferably n.gtoreq.50; more preferably, n.gtoreq.200, still more preferably n.gtoreq.400, and a larger number of cells is more advantageous for improving the accuracy of the result.
The value range of m in the step (1) is preferably that m is more than or equal to 10; more preferably m is not less than 50; still more preferably m.gtoreq.200, still more preferably m.gtoreq.400, and the larger the number of cell domains, the more advantageous the improvement of the accuracy of the results.
In the step (1), taking the data of 2 cell areas as a group, dividing the data into t groups in pairs, wherein the combination of t has at most
For convenience of calculation, two cell areas which are adjacent in sequence can be selected to be combined for calculation; the value range of t is preferably t is more than or equal to 10; more preferably t.gtoreq.50; still more preferably t.gtoreq.200; more preferably, t is more than or equal to 400, and the more times of calculation is, the more the accuracy of the result is improved.
E in step (2)1The value range of (A) is preferably as follows: 0 < e1Less than 0.1; more preferably: e.g. of the type1＝0.05。
The value range of e in the step (2) is preferably as follows: e is more than 0 and less than 0.1; more preferably: e =0.05.
The donor emission spectrum and the acceptor emission filter spectrum, the acceptor excitation spectrum and the acceptor excitation filter spectrum, the acceptor excitation spectrum and the donor excitation filter spectrum, and the relative intensity ratio of the illumination of the donor excitation and the acceptor excitation in the step (2) can be obtained from a device manufacturer or a network open source database.
The solution of the equation in step (2) is preferably an interior point method, and may be obtained by calculation of an auxiliary programming language, such as a programming tool Matlab.
The method for measuring the FRET system correction parameter based on the single standard FRET plasmid is applied to measuring the FRET system correction parameter.
The FRET system correction parameters are FRET system correction parameters beta, lambda, G factors and k factors.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides a technology for measuring correction parameters beta, lambda, G factor and k factor of a FRET system based on a standard FRET plasmid, the technology only needs to image cells transfected with the standard FRET plasmid, at least four experimental samples which are originally needed to be used are reduced to one, the number of biological samples which need to be used is greatly reduced, the experimental efficiency is improved, and the correction parameters of the FRET system are measured from a small amount of biological image data mainly through physical analysis and mathematical calculation. Based on the technology, the correction parameters beta, lambda, G factor and k factor of the FRET system are measured, and the method has high convenience, stability and reliability.
Drawings
FIG. 1 is a graph of the excitation and emission spectra of the donor (Cerulean) and acceptor (Venus) of standard plasmid C32V and the spectra of the filters of the system (Axio Observer 7); wherein, (a) is the excitation spectrum and emission spectrum of the donor, and the spectrum of the acceptor emission filter and the spectrum of the donor emission filter; (b) The spectrum of the acceptor excitation spectrum and the spectrum of the emission spectrum, and the spectrum of the acceptor excitation filter and the spectrum of the donor excitation filter are shown.
FIG. 2 is a diagram of the present invention in which multiple cell regions are combined and solved pairwiseAn exemplary diagram of (a); wherein, (a) is to select 6 cell areas from the three-channel fluorescence intensity image of Hela cells transfected with standard plasmid C32V; (b) Mean fluorescence intensity I for three channels in 6 cell regionsDD、IDA、IAA(ii) a And (c) the result of solving two groups of 6 cell areas in a combined manner.
FIG. 3 is a three-channel fluorescence intensity image of Hela cells transfected with standard plasmid C32V and a normalized histogram of four correction parameters; wherein, (a) is Hela cells transfected with standard plasmid C32V (E)1＝0.31，R1= 1.00) three-channel fluorescence intensity image IDD、IAA、IDA(ii) a And (b) normalized histograms of the four correction parameters.
FIG. 4 is a diagram of a process for verifying the correction parameters of the present invention using standard FRET plasmid C17V; wherein (a) is three-channel fluorescence intensity image I of Hela cells transfected with standard plasmid C17VDD、IAA、IDAAnd an efficiency value EDConcentration ratio to donor/acceptor RC(ii) a (b) Is the efficiency value EDTo donor/acceptor concentration ratio RCIs normalized to the histogram.
FIG. 5 is a three-channel fluorescence intensity image of Hela cells transfected with standard plasmid C5V and a normalized histogram of four correction parameters; wherein, (a) is Hela cells transfected with standard plasmid C5V (E)2＝0.46，R1= 1.00) three channel fluorescence intensity image IDD、IAA、IDA(ii) a And (b) normalized histograms of the four correction parameters.
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. Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.
Example 1
1. Standard FRET plasmid
Given donor-acceptor pairs: the donor is Cerulean (abbreviated as C) and the acceptor is Venus (abbreviated as V).
FRET tandem plasmid structure C32V (abbreviation: standard plasmid C32V): a FRET tandem plasmid structure consisting of a 32 amino acid linker linking C and V;
FRET tandem plasmid structure C17V: a FRET tandem plasmid structure formed by linking C and V through a connecting sequence of 17 amino acids;
the source is as follows: FRET tandem plasmids C17V and C32V were purchased from the U.S. Addgene plasmid library (efficiency values for C32V are known: efficiency value E)1＝0.31)。
2. Wide-field spectral microscopic imaging system
Wide field fluorescence microscopes were produced by Calzaisi, germany under the model Axio Observer7. The light source was X-Cite120Q from Excelitas. The objective lens is an oil lens with the magnification of 63 and the numerical aperture of 1.4 (63 multiplied by 1.4 NA), a rotating wheel provided with six cubes (each cube can be provided with an excitation sheet, a light splitting sheet and an emission sheet respectively), and a CCD camera is externally connected. The wavelength of the excitation light is selected by rotating the cube 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. 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 percentTMThe 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 the cells in the culture dish for 2-3 times by using a serum-free DMEM medium or PBS buffer solution, mainly washing off the 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) And (4) for a sample containing a single plasmid in the cells of one culture dish, after the step (3) is completed for 4-6 hours, absorbing the transfection solution, then washing the cells in the culture dish for 2-3 times by using serum-free DMEM medium or PBS, adding DMEM medium containing newborn bovine serum into the culture dish, and culturing for 24-48 hours to obtain the sample for the experiment.
4. Measurement sample
4.1 HeLa cells were transfected with standard FRET plasmid C32V following the plasmid transfection procedure described above in step 3.
4.2 the specific measurement procedure is as follows:
(1) Four correction parameters to be measured are determined:
the four correction parameters are β, λ, G-factor and k-factor. Where β = a-bd and λ = d-ac. In the practical application of FRET system, only the values of (a-bd) and (d-ac) are used, so that the values of a, b, c and d are not required to be measured. This reduces the original need to measure six correction parameters of the FRET system to four correction parameters.
(2) Determining the range of parameters β and λ:
the system correction parameters beta, lambda, G factor and k factor are all numbers larger than zero, and a constraint condition I is obtained: beta is more than 0, lambda is more than 0, G is more than 0, k is more than 0.
For λ = d-ac and β = a-bd, where c and b are numbers approximately equal to zero, i.e., λ and β are approximately equal to d and a. Theoretical values of λ and β can be determined from the physical concepts of d and a, respectively, and given an approximate range of actual values of λ and β. d depends only on the donor emission spectrum, the filter set and the spectral response of the camera; a depends on the acceptor excitation spectrum and also on the ratio of the illumination powers in the two channels [ Coullomb, a., et al. "QuanTI-FRET: a frame for squarating FRET measurements in lifting cells, "Sci Rep10 (1), 1-11 (2020)]. Thus, the approximate range of λ can be determined from d. The maximum efficiency value (e) at the intersection of the spectral passbands of the donor emission spectrum and the acceptor emission filterDa) Maximum efficiency value (e) at intersection with donor emission spectrum and donor emission filter spectral passbandDd) The ratio of (d) is denoted as λ' = eDa/eDd) λ' is the theoretical value of λ, further oneThe second constraint for determining λ is λ '+ e > λ' -e, where e is a constant greater than zero (0 < e < 1), preferably 0.05. Similarly, the approximate range of β is determined by a. The maximum efficiency value (e) at the intersection of the spectral passbands of the acceptor excitation spectrum and the donor excitation filterdA) Maximum efficiency value (e) at intersection with the receptor excitation spectrum and the spectral passband of the receptor excitation filteraA) The resulting value of the ratio of (d) to (d) multiplied by the relative intensity ratio phi of the illumination of the donor excitation and the acceptor excitation is denoted as beta' = (e)dA/eaA) Phi), beta 'is the theoretical value of beta, and a second constraint for further determining beta is beta' + e1＞β＞β′-e1Wherein e is1Is a constant greater than zero (0 < e)1< 1), preferably 0.05.
The wide-field spectral microscopic imaging system in the experiment adopts a BP436/20 excitation filter and a BP500/20 excitation filter which respectively correspond to donor excitation and acceptor excitation, and adopts a BP480/40 emission filter and a BP535/30 emission filter which respectively correspond to donor emission and acceptor emission. The excitation and emission spectra of the imaging system and of the donor (Cerulean) and acceptor (Venus) of the standard plasmid C32V used for imaging are shown in fig. 1.
As shown in fig. 1 (a), the ratio λ' of the maximum efficiency value at the intersection of the donor emission spectrum and the acceptor emission filter BP535/30 passband to the maximum efficiency value of 1.000 at the intersection of the donor emission spectrum and the donor emission filter passband is 0.611, plus or minus 0.05 determines the range of λ, giving the λ second constraint: 0.661 & gtlambda & gt 0.561.
As shown in fig. 1 (b), the ratio of the maximum efficiency value of 0.054 at the intersection of the acceptor excitation spectrum and the passband of the donor excitation filter BP436/20 to the maximum efficiency value of 0.902 at the intersection of the acceptor excitation spectrum and the passband of the acceptor excitation filter is multiplied by the illumination relative intensity ratio phi of the donor excitation to the acceptor excitation (the relative intensity ratio of the light source X-Cite120Q at the wavelength 446nm to 510nm is about 2.5), calculated as beta' is 0.150, and the range of beta is determined by adding or subtracting 0.05, to obtain the third constraint of beta: 0.200 & gtbeta & gt 0.100.
(3) Establishing a constrained multivariate equation:
building a beltConstrained multivariate equation (1). The unknowns in the equation are the four correction parameters β, λ, G-factor and k-factor to be measured. Efficiency value E in equation1To donor/acceptor concentration ratio R1Is determined from a standard FRET plasmid selected and is a known value. The constraints (s.t.) in the equation are determined by step (2). In the equation (I)DD1，IAA1，IDA1) And (I)DD2，IAA2，IDA2) Is a three-channel image (I) with background subtractedDD、IAA、IDA) Mean value of fluorescence intensity of 2 selected cell areas.
Wherein:
the efficiency value E of the standard FRET plasmid C32V used in this experiment is known1=0.31, concentration ratio of donor and acceptor R1=1.00, and the constraints determined above. Substituting the above multivariable equation (1) to obtain the multivariable equation (2), which is as follows:
wherein:
(4) Obtaining experimental data, substituting the experimental data into an equation, solving and determining a FRET system correction parameter:
hela cells transfected with standard plasmid C32V (E)1＝0.31，R1= 1.00) three-channel fluorescence imaging is carried out, and a three-channel fluorescence intensity image I is collectedDD、IAA、IDAThe exposure time settings are the same. The following configuration was performed using wide field fluorescence microscopy imaging: i acquisition Using an excitation Filter of Filter set BP436/20, a Beam of DFT 455, and an emission Filter of BP480/40DDImages, using an excitation filter of filter set BP500/20, a beam splitter of DFT 515, and an emission filter of BP535/30 to obtain IAAThe image is acquired by using an excitation filter of a filter set BP436/20, a beam splitter of DFT 455 and an emission filter of BP535/30DAAnd (4) an image. The three-channel fluorescence intensities in this experiment were all mean fluorescence intensities unless otherwise specified.
Each channel image was background subtracted and the cell regions were automatically circled. The larger the number of the selected cell areas, the more favorable the accuracy of the result. Two data were then randomly selected as a set of substitution equations and calculated, and repeated multiple times. The method comprises the following specific steps: as shown in FIG. 2, three-channel fluorescence imaging acquisition I is carried out firstDD、IAA、IDAImage, acquiredThree-channel fluorescence intensity image IDD、IAA、IDAComprises 2 cells, then 6 cell regions are selected in the 2 cells (the experiment is performed in 2 cells, 3 cell regions are selected in each circle (the number in the figure is 1-6), in practical application, more than 1 cell can be selected according to practical requirements, and more than 2 cell regions are selected from the cells), the average fluorescence intensity values of the 6 cell regions are obtained, and then 6 groups of data corresponding to the 6 cell regions are freely combined in pairs (the maximum number is that the 6 groups of data are combined in pairs)
One combination can be selected randomly or more than 2 combinations are selected and then the average value is obtained), the use equation is substituted and solved, and the correction parameters beta, lambda, G and k are obtained through calculation. Wherein, the first and the second end of the pipe are connected with each other,
the equations that are brought into the data are:
min f(β，λ，G，k)＝|y1|+|y2|+|y3|+|y4|
s.t.
β＞0；λ＞0；
G＞0；k＞0；
0.661＞λ＞0.561；
0.200＞β＞0.100；
wherein:
this program automatically circles 404 cell regions from fig. 3 (a) and obtains the mean fluorescence intensity value for each cell region. Three-channel fluorescence intensity values (I) in 404 cell regionsDD、IAA、IDA) (rounding up) is (2019, 4499, 4475), (678, 1534, 1604.)... (2286, 5121, 5146), respectively.
Two data were randomly selected as a set of substitution equations and calculated, repeated multiple times. For example, a set of data values (2019, 4499, 4475) and (678, 1534, 1604) are substituted into the usage equation and solved for.
Solving the equation by using an interior point method (using a programming tool Matlab) to obtain an optimal value of f =0.039 (rounded to the 3 rd digit on the right side of the decimal point), wherein the correction parameters beta, lambda, G and k corresponding to the first group of data are beta1＝0.146，λ1＝0.609，G1＝3.179，k1＝0.641。
And repeating for multiple times. Here, the measurement is repeated 202 times (in total
The seed combination is not limited by cell region selection, two random numbers between 1 and 404 are generated by programming, and two groups of data corresponding to the random numbers are combined and calculated to obtain a result, namely free combination is also feasible; for convenience of calculation, the experiment selects two cell area combinations which are adjacent in sequence for calculation, namely 404 cell areas are selected, then the two data which are adjacent in sequence are freely combined to form a group which is put into the equation and calculated, and 202 groups of solutions (beta, lambda, G, k) are obtained, namely (0.146, 0.609,3.179, 0.641), (0.145, 0.609,3.051, 0.660) are obtained, namely (0.146, 0.610,3.051, 0.659).
The normalized histogram distribution of the four correction parameters is shown in FIG. 3 (b), and the mean value β of the 202 solutions is obtained simultaneouslyv＝0.146，λv＝0.610，Gv＝3.034，kv=0.648, final FRET correction parameter is determined as βv＝0.146，λv＝0.610，Gv＝3.034，kv＝0.648。
5. Authentication
And (3) verifying whether the correction parameters are reliable (accurate) by using standard plasmids, wherein the verification process comprises the following steps:
hela cells were transfected with standard FRET plasmid C17V as described above for plasmid transfection step 3. The correction parameter obtained using the method described above, namely four correction parameters betav＝0.146，λv＝0.610，Gv＝3.034，kv=0.648, efficiency value E of C17V transfected Hela cells was measuredDTo donor/acceptor concentration ratio RCThe measurement results are shown in fig. 4.
According to the formula
And formula
[T.Zal and N.R.J.Gascoigne，“Photobleaching-corrected FRET efficiency imaging of live cells，”Biophys.J.86(6)，3923-3939(2004)；H.Chen et al.，“Measurements of FRET Efficiency and Ratio of Donor to Acceptor Concentration in living Cells，”Biophys.J.91(5)，L39-L41(2006)]Measuring ED＝0.37、RC=0.96. Measurement results and literature reports (YinA, sun H, chen H, liu Z, tang Q, yuan Y, et al. Measuring catalysis factors by imaging a dis of cell expression measurements A2021)DConcentration ratio to donor/acceptor RC(C17V efficiency value EDIs 0.38 +/-0.03 percent of RC1.00 +/-0.05) are kept consistent, the method can be verified to be reliable (accurate).
Example 2
1. Standard FRET plasmid
FRET tandem plasmid structure C5V: a FRET tandem plasmid structure formed by linking C and V through a connecting sequence of 5 amino acids;
the source is as follows: FRET tandem plasmid C5V was purchased from the U.S. Addgene plasmid library (efficiency values for C5V are known: efficiency value E1＝0.46)。
2. Wide-field spectral microscopic imaging system
The same as in example 1.
3. Cell culture and plasmid transfection
The standard FRET plasmid C32V was replaced with the standard FRET plasmid C5V, and the procedure was the same as in example 1.
4. Measurement sample
With reference to the method of example 1, hela cells transfected with standard FRET plasmid C5V (efficiency value E)2=0.46 to donor/acceptor concentration ratio R1= 1.00) three-channel fluorescence imaging is carried out, and a three-channel fluorescence intensity image I is collectedDD、IAA、IDAAs shown in fig. 5 (a).
Selecting 200 cell regions to obtain three-channel fluorescence intensity value (I)DD、IAA、IDA) Substituting the equation to calculate, calculating 100 times by using two data as a group to obtain four correction parameter normalized histograms as shown in FIG. 5 (b), and calculating to obtain a result of βv＝0.145，λv＝0.610，Gv＝3.030，kv＝0.624。
5. Authentication
With reference to the method of example 1, the efficiency value E of C17V transfected Hela cells was measured using standard FRET plasmid C17V to verify whether the correction parameters were reliable (accurate)D=0.36 and donor/acceptor concentration ratio RC=0.96, in agreement with the above literature reports.
The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be equivalent substitutions, such as that the crosstalk coefficients β and λ (β = a-bd, λ = d-ac) can be obtained by solving equations simultaneously, or in practical applications, the crosstalk coefficients can be obtained by calculating the crosstalk coefficients and then solving the equations by simplifying the equations separately transfected donor and receptor samples, and any method for replacing, simplifying, and combining the processes is included in the protection scope of the present invention.
Claims (10)
1. A method for measuring a correction parameter of a FRET system based on a single standard FRET plasmid is characterized by comprising the following steps:
(1) Obtaining three-channel average fluorescence intensity data by circling cell area
Knowing the efficiency value E1Concentration ratio to donor acceptor R1After the transfected plasmid is successfully expressed, n cells are selected to respectively carry out three-channel fluorescence imaging to obtain three-channel average fluorescence intensity IDD、IAA、IDAImages, then at IDD、IAA、IDASelecting m different cell areas in the image, and respectively obtaining three-channel average fluorescence intensity IDD1、IAA1、IDA1，IDD2、IAA2、IDA2......IDDm、IAAm、IDAmAnd the mean fluorescence intensity I of three channels obtained from the 1 st cell regionDD1、IAA1、IDA1Three-channel mean fluorescence intensity I obtained for the 2 nd cell region for the first panel of dataDD2、IAA2、IDA2Three-channel mean fluorescence intensity I obtained for the m-th cell region for the second subset of data, and so onDDm、IAAm、IDAmThe data of the m group is divided into t groups by taking the data of 2 cell areas as a group; wherein n is a positive integer and n is more than or equal to 1, m is more than or equal to 2, t is more than or equal to 1;
(2) Calculating the correction parameters beta, lambda, G factor and k factor of FRET system
(1) Calculating a FRET system correction parameter beta according to a first group of data in the t groups of data obtained in the step (1) and the following equation1，λ1，G1And k1：
min f(β1，λ1，G1，k1)＝|y1|+|y2|+|y3|+|y4|
s.t.
β1＞0；λ1＞0；G1＞0；k1＞0；
λ′+e＞λ1＞λ′-e；
β′+e1＞β1＞β′-e1；
0＜e1＜1；0＜e＜1；
e is a constant greater than zero, e1Is a constant greater than zero;
wherein:
λ' is the maximum efficiency e at the intersection of the donor emission spectrum and the acceptor emission filter spectral passbandDαMaximum efficiency e at the intersection with the donor emission spectrum and the spectral passband of the donor emission filterDdRatio λ' = e of (c) = cDa/eDd；
Beta' is the highest efficiency value e at the intersection of the spectral passbands of the acceptor excitation spectrum and the donor excitation filterdAMaximum efficiency value e at intersection point of receptor excitation spectrum and receptor excitation filter spectral pass bandaAIs multiplied by the resulting value of the relative intensity ratio phi of the illumination of the donor excitation and acceptor excitation, beta' = (e)dA/eaA)*φ；
(2) Replacing the first group of data in the step (1) with the second group of data, and calculating the FRET system correction parameter beta according to the equation2，λ2，G2And k2(ii) a And analogizing until the FRET system correction parameter beta of the t-th group of data is obtained by calculationt，λt，GtAnd kt(ii) a Finally, respectively calculate beta1、β2......βt，λ1、λ2......λt，G1、G2......GtAnd k1、k2......ktAverage value of (beta)v，λv，Gv，kv(ii) a Beta is beta ofv，λv，Gv，kvNamely, the FRET system correction parameters beta, lambda, G factor and k factor.
2. The method of claim 1 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the number ratio of the donor to the acceptor of the standard FRET plasmid in the step (1) is 1: N, and N is more than or equal to 1.
3. The method of claim 2 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the standard FRET plasmid described in step (1) has a donor to acceptor ratio of 1: 1.
4. The method of claim 3 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the standard FRET plasmid in the step (1) is any one of standard FRET plasmids C5V, C17V, C32V and CTV.
5. The method of claim 1 for measuring a FRET system corrective parameter based on a single standard FRET plasmid, wherein:
the value range of n in the step (1) is that n is more than or equal to 10;
the value range of m in the step (1) is that m is more than or equal to 10;
the value range of t in the step (1) is that t is more than or equal to 10;
e in step (2)1The value range of (A) is as follows: 0 < e1＜0.1；
The value range of e in the step (2) is as follows: e is more than 0 and less than 0.1.
6. The method of claim 5 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the value range of n in the step (1) is that n is more than or equal to 50;
the value range of m in the step (1) is that m is more than or equal to 50;
the value range of t in the step (1) is that t is more than or equal to 50;
e in step (2)1The value range is as follows: e.g. of a cylinder1＝0.05；
The value range of e in the step (2) is as follows: e =0.05.
7. The method of claim 6 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the value range of n in the step (1) is that n is more than or equal to 200;
the value range of m in the step (1) is that m is more than or equal to 200;
the value range of t in the step (1) is that t is more than or equal to 200.
8. The method of claim 1 for measuring a FRET system corrective parameter based on a single standard FRET plasmid, wherein:
the three-channel average fluorescence intensity in the step (1) is the three-channel average fluorescence intensity after background subtraction.
9. The method of claim 1 for measuring FRET system correction parameters based on a single standard FRET plasmid, wherein:
the cells in the step (1) are Hela cells;
the solution of the equation in step (2) is an interior point method.
10. Use of a method for measuring FRET system corrective parameters based on a single standard FRET plasmid according to any of claims 1 to 9 for measuring FRET system corrective parameters, wherein:
the FRET system correction parameters are FRET system correction parameters beta, lambda, G factors and k factors.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111083669.8A CN113899722B (en) | 2021-09-14 | 2021-09-14 | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111083669.8A CN113899722B (en) | 2021-09-14 | 2021-09-14 | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113899722A CN113899722A (en) | 2022-01-07 |
| CN113899722B true CN113899722B (en) | 2022-11-01 |
Family
ID=79028461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111083669.8A Active CN113899722B (en) | 2021-09-14 | 2021-09-14 | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113899722B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002085667A2 (en) * | 2001-02-16 | 2002-10-31 | The Johns Hopkins University School Of Medicine | Three cube fret method (33-fret) for detecting fluorescence energy transfer |
| CN105466902A (en) * | 2016-01-18 | 2016-04-06 | 华南师范大学 | Method for measuring fluorescence resonance energy transfer sensitized quenching conversion factors |
| CN106442455A (en) * | 2016-11-23 | 2017-02-22 | 华南师范大学 | Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection |
| CN112129737A (en) * | 2020-09-24 | 2020-12-25 | 华南师范大学 | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof |
| CN113049555A (en) * | 2021-03-12 | 2021-06-29 | 师大瑞利光电科技(清远)有限公司 | Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200232020A1 (en) * | 2019-01-22 | 2020-07-23 | Canon Virginia, Inc. | Partition-free digital PCR (dPCR) system |
-
2021
- 2021-09-14 CN CN202111083669.8A patent/CN113899722B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002085667A2 (en) * | 2001-02-16 | 2002-10-31 | The Johns Hopkins University School Of Medicine | Three cube fret method (33-fret) for detecting fluorescence energy transfer |
| CN105466902A (en) * | 2016-01-18 | 2016-04-06 | 华南师范大学 | Method for measuring fluorescence resonance energy transfer sensitized quenching conversion factors |
| CN106442455A (en) * | 2016-11-23 | 2017-02-22 | 华南师范大学 | Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection |
| CN112129737A (en) * | 2020-09-24 | 2020-12-25 | 华南师范大学 | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof |
| CN113049555A (en) * | 2021-03-12 | 2021-06-29 | 师大瑞利光电科技(清远)有限公司 | Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application |
Non-Patent Citations (2)
| Title |
|---|
| Automated E-FRET microscope for dynamical live-cell FRET imaging;C. ZHANG et al.;《Journal ofMicroscopy》;20191231;第274卷(第1期);第45-54页 * |
| 基于单波长激发的定量FRET成像;魏丽春 等;《激光生物学报》;20180831;第27卷(第4期);第326-331页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113899722A (en) | 2022-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Thevathasan et al. | Nuclear pores as versatile reference standards for quantitative superresolution microscopy | |
| CN112129737B (en) | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof | |
| CN106442455B (en) | A kind of method for fast measuring of the transferring efficiency of fluorescence resonance energy detected simultaneously based on binary channels fluorescence intensity | |
| Żurek-Biesiada et al. | Localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution | |
| CN113049555B (en) | Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application | |
| CN113899722B (en) | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof | |
| CN105466902A (en) | Method for measuring fluorescence resonance energy transfer sensitized quenching conversion factors | |
| CN106290268A (en) | A kind of method utilizing single series connection donor-acceptor structure measurement receptor donor ratio of extinction coefficient | |
| Jiang et al. | Predicting the conformations of the silk protein through deep learning | |
| CN109946278A (en) | Fluorescent dye DAPI carries out cell DNA to dye quantitative screening for cancer and diagnostic method | |
| CN108491688B (en) | Method for preprocessing FRET (fluorescence resonance energy transfer) double-hybridization detection data based on donor-acceptor concentration ratio | |
| CN113960001B (en) | AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof | |
| CN106706587A (en) | FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of excitation spectrum and emission spectrum | |
| CN212008213U (en) | Synchronous fluorescence detection device for interface concentration of fluorescent pollutants | |
| CN102636465A (en) | FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation | |
| CN110376190B (en) | Spectrum-based cell culture suspension pH value detection method | |
| CN107271422B (en) | A method of it is separated based on excitation-emission spectrum while measuring the ratio between the ratio between Acceptor-Donor quantum yield and extinction coefficient | |
| Yan et al. | [24] Fluorescence resonance energy transfer imaging microscopy and fluorescence polarization imaging microscopy | |
| Zucker et al. | Wavelength and alignment tests for confocal spectral imaging systems | |
| Spriet et al. | Correlated fluorescence lifetime and spectral measurements in living cells | |
| KASTEN | Stability of the Feulgen-deoxyribonucleic acid absorption curve in situ with variation in nuclear protein content and other factors | |
| Szikora et al. | Superresolution microscopy of drosophila indirect flight muscle sarcomeres | |
| Galbraith et al. | Multiple spectral parameter imaging in quantitative fluorescence microscopy. I: Quantitativn of bead standards | |
| Caspersson | Quantitative cytochemical determinations on endonuclear structures | |
| CN109916871B (en) | FRET efficiency quantitative measurement method applied to apoptosis detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |