CN112129737A - Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof - Google Patents
Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof Download PDFInfo
- Publication number
- CN112129737A CN112129737A CN202011012673.0A CN202011012673A CN112129737A CN 112129737 A CN112129737 A CN 112129737A CN 202011012673 A CN202011012673 A CN 202011012673A CN 112129737 A CN112129737 A CN 112129737A
- Authority
- CN
- China
- Prior art keywords
- cells
- fret
- ratio
- plasmid
- donor
- 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.)
- Granted
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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for simultaneously and automatically measuring a FRET system correction parameter and a donor extinction coefficient ratio and application thereof. Firstly, using a crosstalk correction sample to obtain crosstalk coefficients a, b, c and d, then respectively transfecting cells with different FRET plasmids independently, carrying out E-FRET imaging, and respectively determining ratio ranges R of the cells transfected with the different plasmids; then, after the cells are transfected separately by various FRET plasmids, carrying out mixed culture and E-FRET imaging to obtain the ratio R of the corresponding cells and carrying out cell classification; and finally, calculating or fitting according to the cell classification and the corresponding E-FRET imaging data to obtain a G factor, a k factor and an extinction coefficient ratio gamma. The method can carry out cell segmentation on the cell image acquired in real time and cell classification for expressing different plasmids, and obtains G factors, k factors and extinction coefficient ratios gamma according to the cell classification, thereby having high 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 simultaneously and automatically measuring a FRET system correction parameter and an extinction coefficient ratio of a donor and an acceptor 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 by donor excitation light), acceptor channel image (I)AAAcceptor fluorescence detected in the acceptor fluorescence detection channel upon excitation by 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 (G factor and k factor) and the extinction coefficient ratio (gamma) of the receptor are key parameters for quantitative FRET detection [ Zal T, Gascoigne N R J].Biophysical journal,2004,6(6):3923-3939；Butz E S,Ben-Johny M,Shen M,et al.Quantifying macromolecular interactions in living cells using FRET two-hybrid assays[J].Nature protocols,2016,11(12):2470-2498；Ben-Johny M,Yue DN,Yue DT.Detecting stoichiometry of macromolecular complexes in live cells using FRET[J].Nature communications,2016,7(1):1-0]. 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 without FRET; extinction coefficient ratio gamma refers to the ratio of the extinction coefficients of the acceptor-donor at the excitation wavelength of the donor [YFP(λex,D)/CFP(λex,D) [ MEANS FOR solving PROBLEMS ] is provided. Correction parameters and extinction system for a given FRET fluorophore pair and imaging systemThe ratio is a constant. The measurement to obtain reliable G factor, k factor and extinction coefficient ratio gamma is the key of quantitative FRET measurement.
There are several methods for measuring factor G. Zal and Gascoigne [ T.Zal and N.R.J.Gascoigne, "Photostabilized FRET interference of live cells," Biophys.J.86(6), 3923-. Nagy et al [ Nagy, Peter, et al. "Novel catalysis method for flow Cytometry luminescence emission reaction between visible luminescence proteins," Cytometry Part a 67(2)86-96(2005) ] determine the G value by three FRET tandem structures with donor-acceptor concentration ratio of 1:1 and different E values, which requires many samples of living cells, and is complicated in experimental process and difficult in data processing. 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 determining factor G in living Cells using two FRET tandem structures with a 1:1 Ratio of Acceptor concentrations and unknown E values, which requires the preparation of two FRET living cell samples of different efficiencies, and both FRET samples must be measured under identical conditions. The group [ J.Zhang et al, "Reliable measurement of the FRET sensitive-quantification factor for FRET quantification in the cells," Micron 88,7-15 (2016.) ] proposes a method of measuring the G value by placing two cells transfected with different plasmids in the same cell culture dish, overcomes the limitation of keeping the same measurement conditions for the two dishes of cells, and improves the stability of the G value measurement. The method firstly ensures the premise that cells expressing two FRET plasmids are measured under the same condition, secondly only needs to carry out data acquisition on one cell, not only improves the experimental efficiency, but also becomes possible for developing the measurement technology of the on-line system correction parameters and the extinction coefficient ratio of the donor and the acceptor. The disadvantages caused by the above are also existed, and how to distinguish the cells expressing different plasmids is a difficult problem to be solved urgently. Theoretically, the larger the difference between the FRET efficiencies of different plasmids is, the more accurate the measured G factor is, but the FRET efficiency is relatively lower or the FRET efficiencies of different expressed plasmids are relatively close, so that signals are easily buried in other signals and are not easy to distinguish, and the condition of incomplete data is caused.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for simultaneously and automatically measuring the correction parameters of a FRET system and the extinction coefficient ratio of a donor and a receptor.
Another object of the present invention is to provide the application of the method for simultaneously and automatically measuring the correction parameter and the extinction coefficient ratio of the FRET system.
The purpose of the invention is realized by the following technical scheme:
a method for simultaneously and automatically measuring the correction parameter of a FRET system and the extinction coefficient ratio of a donor and a receiver comprises the following steps:
(1) and (3) measuring a crosstalk coefficient:
respectively transfecting cells with donor plasmids and acceptor plasmids (crosstalk correction samples), and respectively carrying out E-FRET imaging to obtain three-channel average fluorescence intensity IDD、IAA、IDAAnd calculating to obtain crosstalk coefficients a, b, c and d;
(2) measurement of F of cells transfected with FRET plasmid aloneCi-N/IDDi-NRange of ratio values:
respectively transfecting cells with the I FRET plasmids with different efficiencies, after the cells adhere to the wall, respectively selecting more than N cells or regions from the cells transfected with each plasmid for E-FRET imaging, and respectively obtaining three-channel average fluorescence intensity I of the cells or regions transfected with the 1 st plasmidDD1-1、IDD1-2……IDD1-N，IAA1-1、IAA1-2……IAA1-N，IDA1-1、IDA1-2……IDA1-NAnd calculating the acceptor-sensitized fluorescence intensity FC1-1、FC1-2……FC1-NThen calculating the ratio R as R1-1＝FC1-1/IDD1-1、R1-2＝FC1-2/IDD1-2……R1-N＝FC1-N/IDD1-NThen, according to the total ratio R of more than N cells or regions transfected with the 1 st plasmid, taking 0.01-0.1 (preferably 0.1) as interval to make statistical histogram, and according to the result, selecting the half-height width of peak (full width at half maximum, half-peak width) as the ratio range R of the cells transfected with the 1 st plasmid1Or adding or subtracting 0.01-0.1 as a ratio range R on the basis of the R value corresponding to the peak value of the statistical histogram1Or determining the ratio range R from the maximum and minimum values1(ii) a Then adopting the same method to respectively obtain the ratio range of cells from 2 nd plasmid to i th plasmid as R2、R3……Ri(ii) a Wherein i is not less than 2 and is an integer; n is not less than 20 and is an integer;
(3) measurement of F in Mixed culture of multiple FRET plasmid Single-Trans cellsCi′/IDDi' ratio value:
respectively and independently transfecting cells with the FRET plasmids in the step (2), merging the cells expressing different FRET plasmids into the same culture dish for culture after the transfected plasmids are successfully expressed, selecting more than m cells or regions for E-FRET imaging after the cells are attached to the wall, and respectively obtaining three-channel average fluorescence intensity IDD1′、IDD2′……IDDm′，IAA1′、IAA2′……IAAm' and IDA1′、IDA2′……IDAm', and calculating the acceptor-sensitized fluorescence intensity F according to the crosstalk coefficients a, b, c and d obtained in the step (1)C1′、FC2′……FCm', calculating the ratio R1′＝FC1′/IDD1′、R2′＝FC2′/IDD2′……Rm′＝FCm′/IDDm'; wherein m is more than or equal to 5;
(4) cell classification:
according to R obtained in the step (3)1′、R2′……Rm' size, classifying its corresponding cell into the cell type of step (2): such as R1' at R1Within a range of (i.e. R)1' corresponding cells are cells transfected with the 1 st plasmid, e.g.R1' at R2Within a range of (i.e. R)1' corresponding cells are cells transfected with the 2 nd plasmid, e.g.R1' at RiWithin a range of (i.e. R)1' the corresponding cells are cells transfected with the i plasmid; by analogy, respectively combine R2′……Rm' performing classification;
(5) calculating or fitting to obtain a G factor, a k factor and an extinction coefficient ratio gamma:
selecting average fluorescence intensity and receptor-sensitized fluorescence intensity of cells corresponding to at least 2 plasmids from the I plasmids according to the classification in the step (4) and the data obtained in the step (3), and marking as IDD1″、IDD2″……IDDi″，IAA1″、IAA2″……IAAi″，FC1″、FC2″……FCi", y is calculated for cells or regions of various transfected plasmids1＝FC1″/(a×IAA1") and x1＝IDD1″/(a×IAA1″)，y2＝FC2″/(a×IAA2") and x2＝IDD2″/(a×IAA2″)……yi＝FCi″/(a×IAAi") and xi＝IDDi″/(a×IAAi") and then calculating the G factor, the k factor and the extinction coefficient ratio gamma according to the calculation result, and dividing the calculation result into the following two cases:
when i is 2: g ═ y2-y1)/(x1-x2) (ii) a k is the average value of the k-factor for each cell or region; γ ═ a/(G × k);
when i is>At 2, will be x1，x2……xiAs the abscissa, in y1，y2……yiAnd (3) obtaining a straight line through least square fitting as a vertical coordinate, wherein the absolute value of the slope of the straight line is a G factor, the reciprocal of the y-axis intercept of the straight line is an extinction coefficient ratio gamma, and finally calculating a k factor, namely the average value of the k factor of each cell or area.
The method for simultaneously and automatically measuring the ratio of the correction parameter of the FRET system to the extinction coefficient of the donor and the acceptor comprises the steps of (1) to (3) carrying out E-FRET imaging, carrying out cell or region segmentation on three channel images when measuring the fluorescence intensity of the donor, the fluorescence intensity of the acceptor and the FRET, and carrying out background subtraction by taking a cell-free region as a background.
The donor plasmid and the acceptor plasmid in the step (1) are used as crosstalk correction samples for measuring crosstalk coefficients, namely, cells singly transfected with the donor plasmid are used for measuring crosstalk coefficients c and d, and cells singly transfected with the acceptor plasmid are used for measuring crosstalk coefficients a and b; preferred are Cerulean (C) and Venus (V), available from the Addgene plasmid library, USA.
The types of i in the step (2) are 2-4; preferably 4.
The FRET plasmids in the step (2) can be constructed to obtain FRET plasmids with different FRET efficiencies by adjusting the distance between the donor and the acceptor; 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.
In the step (2), since the expression levels of the cells transfected with different plasmids are different, E-FRET imaging needs to be performed on the cells transfected with different plasmids for early division of RiValue range (R)iIs a range value).
The region in step (2) may be defined as the actual case may be, and may be the whole cell or a part of the cell.
The value range of N in the step (2) is preferably that N is more than or equal to 50 (the larger the number of cells or regions is, the better the N is).
The selection of the cells described in step (2) can be achieved by: the culture dish of the cells containing the transfection plasmid is placed under a wide field fluorescence microscope, more than 10 different fields are selected, then 1-3 (or 2-3) cells or regions are selected in each field for determination, and the number of the determined cells or regions is generally not less than 20 (preferably not less than 50 cells or regions).
The full width at half maximum of the peak value described in step (2) is preferably obtained by: r to be obtained1-1、R1-2……R1-NAnd counting at intervals of 0.01-0.1, making a histogram, recording the highest frequency of the occurrence of the ratio R as M, and then taking the closed interval of the left and right R value intervals corresponding to the M/2 position as the full width at half maximum of the peak value.
The interval can be set according to actual conditions, and is preferably 0.1.
Determining ratio range R according to the maximum value and the minimum value in step (2)1It is suitable for plasmids with larger difference of FRET efficiency.
The value range of m in the step (3) is preferably that m is more than or equal to 8; more preferably, m.gtoreq.10.
The E-FRET imaging described in steps (1), (2) and (3) is based on FRET microscopy combined by three filters, and quantitative measurement results can be obtained by one-key operation.
In the step (3), when E-FRET imaging is carried out, a plurality of images of different fields can be obtained at one time, and then the three-channel average fluorescence intensity I of the cells under different fields is respectively measuredDD、IAA、IDAThen, the ratio R is calculated.
The cells described in step (4) were classified as cells for differentiating between transfected with different plasmids.
The k factor in the step (5) is calculated by the method as follows: calculating the k factor k for each cell or region separately1＝(IDD1″+FC1″/G)/IAA1″、k2＝(IDD2″+FC2″/G)/IAA2″……ki＝(IDDi″+FCi″/G)/IAAi", and then averaged.
The method for simultaneously and automatically measuring the correction parameter of the FRET system and the extinction coefficient ratio of the donor and the acceptor is applied to the E-FRET detection.
The basic principle of the invention is as follows:
the invention is based on the double-plasmid/multi-plasmid based G factor determination method (named mTP-G) [ Zhang J, Zhang L, Chai L, et al. reliable measurement factor of the FRET sensitive-resonance factor for FRET quantification in living cells [ J ] Micron,2016,88:7-15 ] provided by the group, and can realize rapid and reliable determination of G factor. The k-factor and extinction coefficient ratio γ measurements are based on the following references [ T.Zal and N.R.J.Gascoigne, "Photobaric-corrected FRET interference of live cells," Biophys.J.86(6), 3923-3939 (2004), respectively; chen et al, "Measurements of FRET Efficiency and Ratio of Donor to Acceptor control in living Cells," biophysis.J.91 (5), L39-L41 (2006); bunz E S, Ben-Johny M, Shen M, et al.Quantifying macromolecular interactions in living cells using FRET two-hybrid assays [ J ]. Nature protocols,2016,11(12): 2470-2498; Ben-Johny M, Yue DN, Yue DT. detecting and stoichimetry of macromolecular compounds in living cells using FRET [ J ]. Nature communications,2016,7(1):1-0].
The specific theoretical formula is as follows:
Fc＝IDA-a(IAA-cIDD)-d(IDD-bIAA) (1)
wherein n is the donor to acceptor concentration ratio (the donor to acceptor concentration ratios of the standard plasmids C5V, C17V, C32V and CTV in the present invention are all 1: 1).
Wherein four spectral crosstalk coefficients are measured with cells transfected with Cerulean (abbreviated C) and Venus (abbreviated V), respectively:
a＝IDA(A)/IAA(A) (5)
b＝IDD(A)/IAA(A) (6)
c＝IAA(D)/IDD(D) (7)
d＝IDA(D)/IDD(D) (8)
a is acceptor excitation crosstalk coefficient, b is acceptor emission crosstalk coefficient, c is donor excitation crosstalk coefficient, and d is donor emission crosstalk coefficient; a is a donor and D is an acceptor.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides a technology for automatically measuring G factor, k factor and extinction coefficient ratio gamma on line by using the same dish of cells expressing different donor/acceptor FRET plasmids. The technology can not only carry out cell segmentation and cell classification expressing different plasmids on cell images acquired in real time, but also automatically measure G factors, k factors and extinction coefficient ratios gamma based on the automatic classification of the cell types. Based on the technology, the G factor, the k factor and the extinction coefficient ratio gamma of the one-button automatic online measurement system can be realized, and the stability and the reliability are high.
Drawings
FIG. 1 is a statistical graph of R values of two plasmids obtained by processing samples expressing CTV and C17V plasmids in a dish in an imaging field according to different cells (in the graph, 5 different regions and 1 background region of different cells are circled by using a circled region method, and finally, an average gray value of each region is obtained and is used for calculating related intermediate quantity and final result).
FIG. 2 is an image of a sample expressing four plasmids of CTV, C5V, C17V and C32V, respectively, in a dish, a statistical graph of R values, and a line graph obtained by least squares fitting; wherein, a is the imaging field of a sample in a dish, wherein the sample respectively expresses four plasmids of CTV, C5V, C17V and C32V (in the figure, 1-9 are 8 different regions and 1 background region which are circled by a circled region method, and finally the average gray value of each region is obtained and is used for calculating related intermediate quantity and final result); b is a statistical chart of R values of different cell regions distinguished according to different plasmids; the graph c shows data of different plasmids obtained from the visual field cells, and a straight line obtained by least square fitting can be used to calculate the G factor, the k factor and the extinction coefficient ratio gamma by fitting.
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 present invention are commercially available.
Example 1
1. Plasmids
Given donor-acceptor pairs: the donor is Cerulean (abbreviated as C) and the acceptor is Venus (abbreviated as V).
FRET tandem plasmid structure C5V: a FRET tandem plasmid structure consisting of a 5 amino acid linker linking C and V.
FRET tandem plasmid structure C17V: a FRET tandem plasmid structure consisting of a 17 amino acid linker linking C and V.
FRET tandem plasmid structure C32V: a FRET tandem plasmid structure consisting of a 32 amino acid linker linking C and V.
FRET tandem plasmid structure CTV: a FRET tandem plasmid construct consisting of a 229 amino acid linker linking C and V.
The source is as follows: the donor plasmids Cerulean (C), acceptor plasmids Venus (V), C5V, C32V and FRET reference plasmids C17V and CTV are purchased from the Addgene plasmid library of America.
2. Wide-field spectral microscopic imaging system
Wide field fluorescence microscopes were produced by Karl Zeiss, Germany, under the model Axio Observer 7. The light source is X-Cite120Q from Lumen Dynamics. The objective lens is an oil lens with the magnification of 40 and the numerical aperture of 1.3(40 multiplied by 1.3NA), 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 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 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) and (3) 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 the DMEM medium containing newborn bovine serum into the culture dish, and culturing for 24-48 hours to obtain the sample for the experiment.
(5) For a sample containing multiple plasmids in cells of a culture dish, the preparation method comprises the following steps: after completing the step (3) for 4-6 hours, respectively sucking out transfection liquid from cells of required individual transfection plasmids, cleaning the cells in the culture dish for 2-3 times by using serum-free DMEM culture medium or PBS, adding DMEM culture medium containing newborn bovine serum into the culture dish, digesting the cells transfected with different plasmids in different culture dishes by using 100 mu L pancreatin cell digestive juice after culturing for 16-40 hours, sucking away the digestive juice, slightly blowing the cells for 2-3 times by using 100 mu L serum-free DMEM culture medium, putting cell suspension blown from different culture dishes into the same EP tube, uniformly mixing for 2-3 times, and putting the cell suspension into a new culture dish again, wherein the proportion of the DMEM culture medium to the fetal bovine serum is 10: 1 (volume ratio), adding fetal calf serum, and culturing for 8 hours.
4. Measurement sample
4.1 separately transfecting the C sample, the V sample and the FRET tandem reference sample CTV, C5V, C17V and C32V into Hela cells according to the plasmid transfection steps, wherein 8 hours are required before measuring a sample containing multiple plasmids in the cells of one culture dish, and the cells transfected with different plasmids are digested and mixed into one culture dish. In this experiment, two crosstalk coefficient correction samples, four single-transition FRET tandem reference samples and two experimental samples were prepared as follows:
crosstalk correction sample 1: the C samples were transfected separately and used to measure the crosstalk coefficients C, d.
Crosstalk correction sample 2: v samples were transfected separately and used to measure the crosstalk coefficients a, b.
CTV reference sample: independent transfection of CTV samples for R measurementCTV(R is acceptor-sensitized fluorescence intensity FCRatio to donor fluorescence intensity).
C5V reference sample: C5V samples were transfected individually for R measurementC5VThe range of (1).
C17V reference sample: C17V samples were transfected individually for R measurementC17VThe range of (1).
C32V reference sample: C32V samples were transfected individually for R measurementC32VThe range of (1).
Experimental sample 1: the CTV + C17V double plasmid sample is prepared by digesting and mixing CTV transfected cells and C17V transfected cells into a culture dish.
Experimental sample 2: four plasmid samples of CTV + C5V + C17V + C32V; namely, cells transfected with CTV alone, cells transfected with C5V alone, cells transfected with C17V alone and cells transfected with C32V alone were digested and mixed in a petri dish.
4.2 the specific measurement procedure is as follows:
the wide field fluorescence microscope was configured as follows: light of 436 +/-12.5 nm and light of 510 +/-8.5 nm are respectively used as excitation light of Cerulean and Venus, and channels of 480 +/-11 nm and light of 530 +/-15 nm are respectively used as detection channels of Cerulean and Venus fluorescence.
Note: the three-channel fluorescence intensities in this experiment were all mean fluorescence intensities unless otherwise specified.
(1) And (3) carrying out crosstalk coefficient measurement by using the crosstalk coefficient correction sample:
three-channel fluorescence imaging is respectively carried out by using the Hela cells (namely crosstalk correction sample 1) transfected with C alone and the Hela cells (namely crosstalk correction sample 2) transfected with V alone, and donor fluorescence intensity I is collectedDDAcceptor fluorescence intensity IAAFRET fluorescence intensity (fluorescence detected in the acceptor fluorescence detection channel upon excitation by donor excitation light) IDAThree-channel mean fluorescence intensity (I)DD C＝0.78730、IDA C＝0.48561、IAA C＝0.00378；IDD V＝0.0530、IDA V＝0.6666、IAA V4.8164), and the crosstalk coefficient a is 0.1384, b is 0.0110, c is 0.0048, and d is 0.6168, which are calculated according to the equations (5) to (8).
(2) The measurement reference sample CTV, the reference sample C5V, the reference sample C17V, and the reference sample C32V:
carrying out three-channel fluorescence imaging on cells transfected with different reference samples respectively, and collecting fluorescence intensity I of donorDDAcceptor fluorescence intensity IAAFluorescence intensity of FRETDAThree channels mean fluorescence intensity (I respectively)DD CTV＝1.796、IDA CTV＝1.976、IAA CTV＝3.433；IDD C5V＝0.306、IDA C5V＝1.143、IAA C5V＝1.069；IDD C17V＝1.438、IDA C17V＝4.143、IAA C17V＝4.314；IDD C32V＝2.140、IDA C32V＝4.690、IAA C32V5.302), based on the mean fluorescence intensity and step of the three channelsThe crosstalk coefficients a, b, c, d calculated in step (1) and formula (1) are taken out of a partial region of the cell to calculate the fluorescence intensity F for sensitizing FRET acceptorC(FC CTV＝0.418、FC C5V＝0.814、FC C17V＝2.689、FC C32V2.674), and the acceptor-sensitized fluorescence intensity F was calculatedCRatio of fluorescence intensity of donors R value (R in each case)CTV＝FC CTV/IDD CTV＝0.233、RC5V＝FC C5V/IDD C5V＝2.659、RC17V＝FC C17V/IDD C17V＝1.870、RC32V＝FC C32V/IDD C32V＝1.249)。
Secondly, according to the method of the first step, at least 20 cells or regions (preferably more than 50 cells or regions) are respectively measured on different reference samples, about 20 different fields are selected in the experiment, 2-3 typical cells or regions are selected for each field to be measured, the defined region can be large or small, such as a part of the region in the cells or the region containing the cells), the R value of each cell or region is correspondingly calculated, and then the value range of the R value is determined in the following way: making a histogram of the obtained R values of each cell or region, wherein the histogram is obtained by performing interval division (abscissa) on all the obtained R values at intervals of 0.1 to obtain the frequency of the R values in a corresponding interval (i.e., the number of R values appearing in the interval) (ordinate), recording the highest frequency of the R values as M, and the value range of the R values as a closed interval (i.e., the full width at half maximum of the peak value) of two R value intervals corresponding to M/2, and then selecting the full width at half maximum of the peak value as the value range of the R values according to the statistical result, that is: -0.25. ltoreq.RCTV≤0.25、0.75≤RC32V≤1.25、1.55≤RC17V≤2.05、2.15≤RC5VLess than or equal to 2.65. Of course, other methods can be used to determine the value range of the R value, such as determining the maximum value and the minimum value of the R value of each cell or region obtained as described above (suitable for plasmids with large difference in FRET efficiency, such as the plasmid in this embodiment; for some plasmids with small difference in FRET efficiency, it is easy to cause misclassification during cell classification); the value range of the R value can also be determined in a +/-delta mode, wherein +/-delta is upAnd adding or subtracting 0.01-0.1 on the basis of the corresponding R value of the peak value (highest frequency) to obtain an interval value which is the R value range. In the three methods, different plasmids are selected for subsequent differentiation of different intracellular transfections.
(3) The measurement of the G factor, k factor and extinction coefficient ratio γ was performed for the experimental sample 1 and the experimental sample 2:
carrying out three-channel fluorescence imaging on Hela cells of an experimental sample 1(CTV + C17V dual-plasmid sample), selecting different cells or regions under different fields of view (the more the cells or regions are, the better the cells or regions are, the more the cells orC'and R' values (F)C′/IDD') and taking the average value of R ', and determining the corresponding cell as the cell transfected with the plasmid according to the average value of R ' and the range of the R value in the step (2), wherein the R value can be calculated in a circle of a region in each cell to obtain the R value, then determining which plasmid is transfected in the cell, and then measuring a plurality of circles of regions in the cell to obtain a plurality of groups of data to perform subsequent calculation; if a large amount of data is needed, a plurality of regions with different visual fields in the same culture dish can be circled, corresponding R values are obtained through calculation, then cell classification is carried out, which kind of plasmids are transfected correspondingly by the cells is determined, and finally the data are substituted into a formula for subsequent calculation.
For the experimental sample 1, a field is selected for three-channel fluorescence imaging (as DD, DA, AA in fig. 1 are cellular fluorescence images formed by three channels), regions are circled in the formed images (5 cells and 1 background region are circled), and the R value of each region is calculated to determine which plasmid is transfected in the corresponding region, for example, region 1, region 2, region 3, region 4, region 5, and C17V are transfected. The calculation process is as follows:
(i) calculating the R value of the circled area: the circles calculated here take the mean fluorescence intensity of each of the three channels of region 1 and region 3: i isDD1′＝1.708、IDA1′＝4.961、IAA1′＝5.141；IDD3′＝1.453、IDA3′＝1.377、IAA3' 2.704, and the acceptor-sensitized fluorescence intensity F is calculated from the crosstalk coefficients a, b, c, d calculated in step (1) and equation (1)C1' 3.232 and FC3' -0.126, and then calculating the ratio R1′＝FC1′/IDD1' 1.892 and R3′＝FC3′/IDD3′＝0.087。
(ii) Distinguish which plasmid was transfected in the circled region: according to the range of R' values in step (2): r1′∈RC17V(i.e., R)1' at RC17VIn the range of (1), R3′∈RCTVI.e. R1' corresponding cells are cells transfected with the C17V plasmid, R3' corresponding cells are cells transfected with CTV plasmid.
(iii) Substituting the region data divided by the plasmid into the formula: two kinds of plasmids IDD′、IAA′、FCThe values are substituted into the expressions (2), (3) and (4) to finally obtain the G factor (G ═ 2.84), k factor (k)1＝(IDD1′+FC1′/G)/IAA1′＝0.554，k2＝(IDD3′+FC3′/G)/IAA3' -0.554, taking the average value k of the two equal to 0.554 and the extinction coefficient ratio gamma (gamma equal to 0.088); in general, the k factor is calculated for each cell (or region), and then the average value of all k values is taken, which is the required k factor.
At the same time, calculate y1＝FC1/(a×IAA1) And x1＝IDD1/(a×IAA1) And y is2＝FC2/(a×IAA2) And x2＝IDD2/(a×IAA2) From the calculation results, it was found that when the plasmid type of the sample was 2, the factor G ═ y (y)2-y1)/(x1-x2) (G ═ 2.84), and the k factor and extinction coefficient ratio γ can be calculated from equations (3) and (4).
FIG. 1 is a statistical plot of the R values of different cells obtained in an imaging field in a dish for samples expressing CTV and C17V plasmids, respectively.
② selecting three-channel fluorescence imaging (as shown in figure 2, DD, DA and AA are cell fluorescence images formed by three channels, and normalizing the data obtained by the circle region by deducting background and exposure time to obtain the fluorescence intensity of the three channels) for Hela cells of an experimental sample 2(CTV + C5V + C17V + C32V), circling the region (the region with 8 cells and 1 background region circled therein) in the formed image, calculating the R value of each region to determine which region transfects plasmids, wherein calculating to obtain the regions with CTV in regions 1, 2 and 3, and C5V, 6 and 7, and C17V, and C4 and 5, and C32V, and selecting the data of the three-channel region 1, 4, 6 and four groups of three-channel region 8 after distinguishing the circled regions according to the types of plasmids, C5V, C17V and C32V, the mean fluorescence intensity data for each channel in each group is as follows: i isDD1″＝2.825、IDA1″＝2.948、IAA1″＝5.186；IDD4″＝2.227、IDA4″＝4.778、IAA4″＝5.488；IDD6″＝0.290、IDA6″＝0.732、IAA6″＝0.783；IDD8″＝0.188、IDA8″＝0.598、IAA8And ″ -, 0.577, and the acceptor-sensitized fluorescence intensity F is calculated from the crosstalk coefficients a, b, c, d calculated in step (1) and formula (1)C1″＝0.525、FC4″＝2.684、FC6″＝0.451、FC80.406, and then calculate the ratio R1″＝FC1″/IDD1″＝0.186、R4″＝FC4″/IDD4″＝1.205、R6″＝FC6″/IDD6″＝1.556、R8″＝FC8″/IDD8And 2.161. Therefore, according to the range of R values in step (2): r1″∈RCTV，R4″∈RC32V，R6″∈RC17V，R8″∈RC5V. Namely R1"corresponding cells are cells transfected with CTV plasmid, R4"corresponding cells are cells transfected with the C32V plasmid, R6"corresponding cells are cells transfected with the C17V plasmid, R8"corresponding cells were cells transfected with the C5V plasmid.
When the ratio R 'of the acceptor-sensitized fluorescence intensity to the donor fluorescence intensity is obtained, several typical cells (the more the number of cells or regions, the better the number of cells or regions is greater than 10) in a plurality of different fields can be selected for measurement in the manner of the above step (2), and then the corresponding cells are determined to be the cells transfected with the plasmids according to R' and the range of the R value in the step (2).
For the above experimental sample 2, y was calculated for the cells of region 1, region 4, region 6 and region 81＝FC1″/(a×IAA1") and x1＝IDD1″/(a×IAA1″)，y4＝FC4″/(a×IAA4") and x4＝IDD4″/(a×IAA4″)，y6＝FC6″/(a×IAA6") and x6＝IDD6″/(a×IAA6″)，y8＝FC8″/(a×IAA8") and x8＝IDD8″/(a×IAA8") mixing x1，x4，x6，x8(abscissa) and y1、y4，y6，y8(ordinate) obtaining a straight line through least square fitting, wherein the absolute value of the slope of the straight line is a G factor, the reciprocal of the y-axis intercept of the straight line is an extinction coefficient ratio gamma, and finally calculating a k factor: k ═ I (I)DD+FC/G)/IAA. I.e. y10.731 and x1＝3.936，y43.533 and x4＝2.932，y64.157 and x6＝2.673，y85.087 and x82.354, in xiIs the abscissa, yiA straight line y obtained by performing least square fitting on the ordinate is-2.7434 x + 11.535; g2.743, γ 1/11.535 0.087, k 0.582 (k)1＝(IDD1″+FC1″/G)/IAA2″＝0.582；k2＝0.584；k3＝0.580；k40.582; k is k1、k2、k3And k4Average value of).
FIG. 2a is a typical field of view in the experimental sample 2, FIG. 2b is a statistical graph of R values for different cellular regions differentiated by the expression of different plasmids, and FIG. 2c is a statistical graph of R values for region 1 (y) in the experimental sample 21，x1) Region 4 (y)4，x4) Region 6 (y)6，x6) And region 8 (y)8，x8) Data of (2) in xiIs the abscissa, yiThe straight line obtained by least squares fitting is performed for the ordinate.
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 equivalent substitutions, such as cell classification by defining R value, R ═ FC/IDDFor the purpose of classification criteria, and in practice, any method that uses a change in the spectral information to distinguish between cells transfected with different plasmids is within the scope of the present invention.
Claims (10)
1. A method for simultaneously and automatically measuring the correction parameter of a FRET system and the extinction coefficient ratio of a donor and a receiver is characterized by comprising the following steps:
(1) and (3) measuring a crosstalk coefficient:
respectively transfecting cells with donor plasmids and acceptor plasmids, and respectively carrying out E-FRET imaging to obtain three-channel average fluorescence intensity IDD、IAA、IDAAnd calculating to obtain crosstalk coefficients a, b, c and d;
(2) measurement of F of cells transfected with FRET plasmid aloneCi-N/IDDi-NRange of ratio values:
respectively transfecting cells with the I FRET plasmids with different efficiencies, after the cells adhere to the wall, respectively selecting more than N cells or regions from the cells transfected with each plasmid for E-FRET imaging, and respectively obtaining three-channel average fluorescence intensity I of the cells or regions transfected with the 1 st plasmidDD1-1、IDD1-2……IDD1-N，IAA1-1、IAA1-2……IAA1-N，IDA1-1、IDA1-2……IDA1-NAnd calculating the acceptor-sensitized fluorescence intensity FC1-1、FC1-2……FC1-NThen calculating the ratio R as R1-1＝FC1-1/IDD1-1、R1-2＝FC1-2/IDD1-2……R1-N＝FC1-N/IDD1-NThen, according to the total ratio R of more than N cells or regions for transfecting the 1 st plasmid, taking 0.01-0.1 as interval intervals to make a statistical histogram, and according to the result, selecting the half-height width of the peak value as the ratio range R of the cells for transfecting the 1 st plasmid1Or adding or subtracting 0.01-0.1 as a ratio range R on the basis of the R value corresponding to the peak value of the statistical histogram1Or determining the ratio range R from the maximum and minimum values1(ii) a Then adopting the same method to respectively obtain the ratio range of cells from 2 nd plasmid to i th plasmid as R2、R3……Ri(ii) a Wherein i is not less than 2 and is an integer; n is not less than 20 and is an integer;
(3) measurement of F in Mixed culture of multiple FRET plasmid Single-Trans cellsCi′/IDD′iThe proportion value is as follows:
respectively and independently transfecting cells with the FRET plasmids in the step (2), merging the cells expressing different FRET plasmids into the same culture dish for culture after the transfected plasmids are successfully expressed, selecting more than m cells or regions for E-FRET imaging after the cells are attached to the wall, and respectively obtaining three-channel average fluorescence intensity IDD1′、IDD2′……IDDm′，IAA1′、IAA2′……IAAm' and IDA1′、IDA2′……IDAm', and calculating the acceptor-sensitized fluorescence intensity F according to the crosstalk coefficients a, b, c and d obtained in the step (1)C1′、FC2′……FCm', calculating the ratio R1′＝FC1′/IDD1′、R2′＝FC2′/IDD2′……Rm′＝FCm′/IDDm'; wherein m is more than or equal to 5;
(4) cell classification:
according to R obtained in the step (3)1′、R2′……Rm' size, classifying its corresponding cell into the cell type of step (2): such as R1' at R1Within a range of (i.e. R)1' corresponding cells are cells transfected with the 1 st plasmid, e.g.R1' at R2Within a range of (i.e. R)1' corresponding cells are cells transfected with the 2 nd plasmid, e.g.R1' at RiWithin a range of (i.e. R)1' the corresponding cells are cells transfected with the i plasmid; by analogy, respectively combine R2′……Rm' performing classification;
(5) calculating or fitting to obtain a G factor, a k factor and an extinction coefficient ratio gamma:
selecting average fluorescence intensity and receptor-sensitized fluorescence intensity of cells corresponding to at least 2 plasmids from the I plasmids according to the classification in the step (4) and the data obtained in the step (3), and marking as IDD1″、IDD2″……IDDi″，IAA1″、IAA2″……IAAi″，FC1″、FC2″……FCi", y is calculated for cells or regions of various transfected plasmids1＝FC1″/(a×IAA1") and x1＝IDD1″/(a×IAA1″)，y2＝FC2″/(a×IAA2") and x2＝IDD2″/(a×IAA2″)……yi＝FCi″/(a×IAAi") and xi＝IDDi″/(a×IAAi") and then calculating the G factor, the k factor and the extinction coefficient ratio gamma according to the calculation result, and dividing the calculation result into the following two cases:
when i is 2: g ═ y2-y1)/(x1-x2) (ii) a k is the average value of the k-factor for each cell or region; γ ═ a/(G × k);
when i is>At 2, will be x1，x2……xiAs the abscissa, in y1，y2……yiAnd (3) obtaining a straight line through least square fitting as a vertical coordinate, wherein the absolute value of the slope of the straight line is a G factor, the reciprocal of the y-axis intercept of the straight line is an extinction coefficient ratio gamma, and finally calculating a k factor, namely the average value of the k factor of each cell or area.
2. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the types of i in the step (2) are 2-4;
the value range of N in the step (2) is that N is more than or equal to 50;
the value range of m in the step (3) is that m is more than or equal to 8.
3. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 2, wherein:
the types of i in the step (2) are 4;
the value range of m in the step (3) is that m is more than or equal to 10.
4. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the number ratio of the FRET plasmid as the donor to the acceptor in the step (2) is 1: n or n: 1, n is not less than 1.
5. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 4, wherein:
the number ratio of the FRET plasmid as the donor to the acceptor in the step (2) is 1:1, in a cell culture medium.
6. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the donor plasmid and the acceptor plasmid in the step (1) are Cerulean and Venus respectively;
the FRET plasmids described in the step (2) are at least two of C5V, C17V, C32V and CTV.
7. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the k factor in the step (5) is calculated by the method as follows: calculating the k factor k for each cell or region separately1＝(IDD1″+FC1″/G)/IAA1″、k2＝(IDD2″+FC2″/G)/IAA2″……ki＝(IDDi″+FCi″/G)/IAAi", and then averaged.
8. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the full width at half maximum of the peak value in the step (2) is obtained by the following method: r to be obtained1-1、R1-2……R1-NAnd counting at intervals of 0.01-0.1, making a histogram, recording the highest frequency of the occurrence of the ratio R as M, and then taking the closed interval of the left and right R value intervals corresponding to the M/2 position as the full width at half maximum of the peak value.
9. The method for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio as claimed in claim 1, wherein:
the selection of the cells in the step (2) is realized by the following way: placing the culture dish filled with the cells of the transfection plasmids under a wide-field fluorescence microscope, selecting more than 10 different fields, and then selecting 1-3 cells or regions in each field for determination, wherein the number of the determined cells or regions is not less than 20.
10. Use of a method according to any one of claims 1 to 9 for simultaneously and automatically measuring the FRET system correction parameter and donor extinction coefficient ratio in E-FRET detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011012673.0A CN112129737B (en) | 2020-09-24 | 2020-09-24 | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011012673.0A CN112129737B (en) | 2020-09-24 | 2020-09-24 | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112129737A true CN112129737A (en) | 2020-12-25 |
| CN112129737B CN112129737B (en) | 2022-03-15 |
Family
ID=73841086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011012673.0A Active CN112129737B (en) | 2020-09-24 | 2020-09-24 | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112129737B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113899722A (en) * | 2021-09-14 | 2022-01-07 | 华南师范大学 | Method for measuring FRET system correction parameters based on single standard FRET plasmid and application thereof |
| CN113960001A (en) * | 2021-10-18 | 2022-01-21 | 华南师范大学 | AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5302836A (en) * | 1992-07-16 | 1994-04-12 | Bernard Siu | High speed image acquisition for microelectronics inspection |
| WO2000008054A1 (en) * | 1998-08-08 | 2000-02-17 | Imperial Cancer Research Technology Limited | Modified green fluorescent protein |
| 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 |
| CN106290268A (en) * | 2016-07-12 | 2017-01-04 | 华南师范大学 | A kind of method utilizing single series connection donor-acceptor structure measurement receptor donor ratio of extinction coefficient |
| US20180238901A1 (en) * | 2017-01-25 | 2018-08-23 | Regents Of The University Of Minnesota | High precision spectral fret assays |
| CN108491688A (en) * | 2018-03-26 | 2018-09-04 | 华南师范大学 | A method of FRET double cross detection datas are pre-processed based on donor-receptor concentration ratio |
-
2020
- 2020-09-24 CN CN202011012673.0A patent/CN112129737B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5302836A (en) * | 1992-07-16 | 1994-04-12 | Bernard Siu | High speed image acquisition for microelectronics inspection |
| WO2000008054A1 (en) * | 1998-08-08 | 2000-02-17 | Imperial Cancer Research Technology Limited | Modified green fluorescent protein |
| 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 |
| CN106290268A (en) * | 2016-07-12 | 2017-01-04 | 华南师范大学 | A kind of method utilizing single series connection donor-acceptor structure measurement receptor donor ratio of extinction coefficient |
| US20180238901A1 (en) * | 2017-01-25 | 2018-08-23 | Regents Of The University Of Minnesota | High precision spectral fret assays |
| CN108491688A (en) * | 2018-03-26 | 2018-09-04 | 华南师范大学 | A method of FRET double cross detection datas are pre-processed based on donor-receptor concentration ratio |
Non-Patent Citations (8)
| Title |
|---|
| HUANMIAN CHEN ET AL.: "Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells", 《BIOPHYSICAL JOURNAL: BIOPHYSICAL LETTERS》 * |
| J . ZHANG ET AL.: "Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction", 《JOURNAL OF MICROSCOPY》 * |
| JIANWEI ZHANG ET AL.: "Spectral measurement of acceptor-to-donor extinction coefficientratio in living cells", 《MICRON》 * |
| TOMASZ ZAL ET AL.: "Photobleaching-Corrected FRET Efficiency Imaging of Live Cells", 《BIOPHYSICAL JOURNAL》 * |
| 孙晗 等: "用于在线E-FRET定量成像的自动背景识别与数据筛选", 《光学学报》 * |
| 尹傲 等: "基于光谱分离的定量FRET检测", 《中国激光》 * |
| 张建伟 等: "荧光共振能量转移(FRET)的定量检测及其应用", 《华南师范大学学报( 自然科学版)》 * |
| 梁丽芳 等: "荧光相关谱测量技术研究", 《激光生物学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113049555B (en) * | 2021-03-12 | 2022-09-30 | 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 |
| CN113899722A (en) * | 2021-09-14 | 2022-01-07 | 华南师范大学 | Method for measuring FRET system correction parameters based on single standard FRET plasmid and application thereof |
| CN113899722B (en) * | 2021-09-14 | 2022-11-01 | 华南师范大学 | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof |
| CN113960001A (en) * | 2021-10-18 | 2022-01-21 | 华南师范大学 | AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112129737B (en) | 2022-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112129737B (en) | Method for simultaneously and automatically measuring FRET system correction parameter and donor/acceptor extinction coefficient ratio and application thereof | |
| EP2439515B1 (en) | Disposable blood collection cuvette with probe composition for cell counting and analysis | |
| RU2126962C1 (en) | Method detecting microorganisms in sample, procedures determining sensitivity of microorganisms to antibiotics and system for realization of such methods and procedures | |
| EP2312943B1 (en) | Method for assessing viable cells and the use of n-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (dacm) or n-(9-acridinyl)maleimide (nam) for assessment of apoptosis. | |
| JP2013513087A (en) | System and method for time-related microscopy of biological organisms | |
| CN109266717A (en) | A kind of method and apparatus by single cell analysis detection bacterium drug resistance | |
| CN113049555B (en) | Method for measuring linear separation and quantification of FRET system correction factor based on same system cell sample and application | |
| CN113960001B (en) | AutoQT-FRET method based on primary imaging measurement system correction factor and application thereof | |
| Lu et al. | A sample-preparation-free, automated, sample-to-answer system for cell counting in human body fluids | |
| 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 | |
| JP4810871B2 (en) | Microorganism detection method | |
| CN106706587B (en) | A kind of FRET quantitative detection modification method separated simultaneously based on excitation spectrum and emission spectrum | |
| CN108491688B (en) | Method for preprocessing FRET (fluorescence resonance energy transfer) double-hybridization detection data based on donor-acceptor concentration ratio | |
| García-Ruano et al. | Fluorescence exclusion–a rapid, accurate and powerful method for measuring yeast cell volume | |
| CN113899722B (en) | Method for measuring FRET system correction parameter based on single standard FRET plasmid and application thereof | |
| Caetano Crowley et al. | Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy | |
| Spector et al. | In vitro microtubule dynamics assays using dark-field Microscopy | |
| Dubois et al. | Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup | |
| EP4332551A1 (en) | Method for quantitatively analyzing fluorescent dyes labeled on extracellular vesicle by using fluorescence correlation spectroscopy, and use thereof | |
| JP2009247331A (en) | Method for detecting microorganism and apparatus for measuring the same | |
| Lanzanò | Back to the Future: Fluorescence Correlation Spectroscopy Moves Back in the Cuvette | |
| ES2893198T3 (en) | A method of quantifying the capacity of individual bacterial cell cultures using culture-independent parameters | |
| Pradeep et al. | LVING Reveals the Intracellular Structure of Cell Growth | |
| Sukumar et al. | High-Throughput Whole-Plate Imaging of Cells for Multiple Biological Applications | |
| Jiang et al. | Urine |
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 |