CN107271422A - A kind of separated based on excitation-emission spectrum measures method of the ratio between the acceptor donor quantum yield with the ratio between extinction coefficient simultaneously - Google Patents
A kind of separated based on excitation-emission spectrum measures method of the ratio between the acceptor donor quantum yield with the ratio between extinction coefficient simultaneously Download PDFInfo
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- 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
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- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The present invention discloses a kind of separated based on excitation-emission spectrum while method of the ratio between the measurement acceptor donor quantum yield with the ratio between extinction coefficient, belongs to FRET detection technique fields.This method comprises the following steps：2 kinds of measurement or the excitation-emission spectrum S of two or more acceptor number is identical from donor number ratio and FRET efficiency is different donor-acceptor series connection sampleDA[i], and by SDA[i]Linear separation, which is carried out, according to three excitation-emission spectrum basic vectors obtains three weight factor WD[i], WA[i]And WS[i]；With WA/WDFor independent variable, WS/WDFor dependent variable, linear fit WA[i]/WD[i]、WS[i]/WD[i], the slope and n of linear equationA/nDProduct inverse be KA/KD, the absolute value of intercept is QA/QD.Measurement process of the present invention is simple, time of measuring is short, measurement result is stable and is applied to different detecting systems.
Description
Technical field
The invention belongs to FRET (FRET) detection technique field, and in particular to one kind is based on excitation-emission
Spectrum separation measures the ratio between Acceptor-Donor quantum yield (Q simultaneouslyA/QD) with the ratio between Acceptor-Donor extinction coefficient (KA/KD) side
Method.
Background technology
FRET microscopies based on fluorescin (FPs) have become studies the important of molecular regulation mechanism in living cells
Instrument.Obtain independent of the quantitative FRET signals of detecting system and FPs expressions be carry out between different experiments room it is academic
The premise for exchanging and comparing.In recent years, we realize a kind of based on excitation-emission light on the spectrum micro imaging system of wide field
The quantitative FRET e measurement technologies (ExEm-spFRET) of separation are composed, acceptor can be overcome to excite crosstalk and donor emission crosstalk simultaneously
[Mengyan Du,et al.“Wide-field microscopic FRET imaging using simultaneous
spectral unmixing of excitation and emission spectra”,Opt.Express 24(14),
16037-16051(2016)]。
The ratio between Acceptor-Donor quantum yield (QA/QD) with the ratio between Acceptor-Donor extinction coefficient (KA/KD) it is many quantitative
Two essential Important Parameters of FRET detection techniques.KA/KDSpectral quality, measuring system excitation channel with excitation source
Transfer function and donor it is relevant with the absorption spectrum of acceptor.Wlodarczyk[Wlodarczyk J,Woehler A,Kobe
F,et al.“Analysis of FRET signals in the presence of free donors and
Acceptors ", Biophysical Journal94 (3), 986-1000 (2008)] et al. existed by measuring Plasmid series sample
Rate of induced polarization when dual wavelength is excited obtains KA/KD, this method requires in all FRET measurement process that system is set and keeps constant.
Zhang[Zhang J,Yang F,Chai L,et al.“Spectral measurement of acceptor-to-donor
Extinction coefficient ratio in living cells ", Micron68 (1), 98-106 (2015)] et al. carry
The sp-ECR methods measurement K gone outA/KDWhen need by Plasmid series sample known to a FRET efficiency.For QA/QDValue,
Most of FRET correlative studys are worth to by the quantum yield for the fluorophor reported in citation.But actually
QA/QDIt is not only relevant with the optical property of donor, acceptor fluorophore, also with the environment and measuring system residing for fluorophor
The spectral response performance of transmission channel is relevant.So, measure simultaneously and accurately Q in given measuring systemA/QDAnd KA/KD's
Value is the precondition of a variety of quantitative FRET detections.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, excitation-emission spectrum is based on it is an object of the invention to provide one kind
Separation measures the ratio between Acceptor-Donor quantum yield (Q simultaneouslyA/QD) with the ratio between Acceptor-Donor extinction coefficient (KA/KD) method.
The donor-acceptor concatenation that this method utilizes 2 kinds or two or more acceptor number is identical from donor number ratio and FRET efficiency is different
Sample measures Q simultaneouslyA/QDAnd KA/KD。
The purpose of the present invention is achieved through the following technical solutions：
Obtain respectively and singly turn donor sample, singly turn acceptor sample and 2 kinds or two or more acceptor number and donor number ratio
The donor-acceptor concatenation sample that value is identical and FRET efficiency is different.Hair is excited by linear separation donor-acceptor concatenation sample
The Q under the conditions of given measuring system can quickly be determined by penetrating spectrumA/QDAnd KA/KDValue, and D-A string need not be measured
Join the FRET efficiency of sample.
One kind is separated based on excitation-emission spectrum and measures the ratio between Acceptor-Donor quantum yield (Q simultaneouslyA/QD) and acceptor-confession
The ratio between body extinction coefficient (KA/KD) method, comprise the following steps：
(1) 2 kinds or two or more acceptor number and donor number ratio (n are measuredA/nD) the identical and different confession of FRET efficiency
Excitation-emission spectrum (the S of body-acceptor concatenation sampleDA[i]), and by SDA[i]Carried out according to three excitation-emission spectrum basic vectors linear
Isolated three weight factor WD[i], WA[i]And WS[i]；Wherein subscript i represents i-th kind of donor-acceptor concatenation sample, i >=2；
(2) W obtained according to step (1)D[i]、WA[i]And WS[i]Calculate WA[i]/WD[i]And WS[i]/WD[i]Value；
(3) with WA/WDFor independent variable, WS/WDFor dependent variable, the W obtained in linear fit step (2)A[i]/WD[i]、WS[i]/
WD[i], the slope and n of linear equationA/nDThe inverse of product be KA/KD, the absolute value of the intercept of linear equation is QA/
QD。
Three excitation-emission spectrum basic vectors described in step (1) are respectively the excitation-emission spectrum basic vector S of donorD, by
The excitation-emission spectrum basic vector S of bodyA, for acceptor be sensitized excitation-emission spectrum basic vector SS；
W described in step (1)D[i]Weight, W shared by donor excitation-emission spectrumA[i]For acceptor excitation-emission spectrum institute
Account for weight, WS[i]For for weight shared by acceptor sensitization excitation-emission spectrum；
Donor-acceptor concatenation sample described in step (1) is applied to the donor-acceptor concatenation sample of all Plasmid series
Sheet, wherein acceptor number and donor number ratio (nA/nD) it is preferably 0.5~3；Most preferably 1：1.
Specifically include following steps：
(1) obtain and singly turn donor sample and singly turn acceptor sample, measure the excitation-emission spectrum basic vector S of donorD, acceptor
Excitation-emission spectrum basic vector SA, and for the excitation-emission spectrum basic vector S of acceptor sensitizationS；
(2) 2 kinds or two or more acceptor number and donor number ratio (n are obtainedA/nD) the identical and different confession of FRET efficiency
Body-acceptor concatenation sample, measures the excitation-emission spectrum (S of donor-acceptor concatenation sampleDA[i]), and by SDA[i]Swash according to three
Send out emission spectrum basic vector and carry out linear separation, obtain three weight factor WD[i], WA[i]And WS[i]；
SDA[i]=WD[i]SD+WS[i]SS+WA[i]SA (2)
(3) W obtained according to step (2)D[i]、WA[i]And WS[i]Calculate WA[i]/WD[i]And WS[i]/WD[i]Value.
(4) with WA/WDFor independent variable, WS/WDThe W obtained for dependent variable linear fit step (3)A[i]/WD[i]、WS[i]/
WD[i], the slope and n of linear equationA/nDThe inverse of product be KA/KD, the absolute value of the intercept of linear equation is QA/
QD.Wherein, WA/WD、WS/WD、KA/KDAnd QA/QDFour meet following relation：
In order to preferably illustrate the present invention, below with a measurement QA/QDAnd KA/KDExample illustrate：
Given donor-acceptor concatenation sample (nA/nD=1)：Donor is Cerulean (abbreviation C), and acceptor is Venus (letters
Claim V).
Donor-acceptor concatenation sample C32V：The D-A for being made up of C and V links the catenation sequence of 32 amino acid
Series connection sample.
Donor-acceptor concatenation sample C17V：The D-A for being made up of C and V links the catenation sequence of 17 amino acid
Series connection sample.
Donor-acceptor concatenation sample CTV：The D-A for being made up of C and V links the catenation sequence of 229 amino acid
Series connection sample.
Specific measurement process is as follows：
(1) individually transfect in the liver cancer cells (HepG2 cells) of people and express donor C and acceptor V, and donor-by
Body series connection sample C32V, C17V and CTV；
(2) with wide field fluorescence microscope and camera measurement C and V PLE and emission spectra.Select 436 ± 10nm and 470
± 10nm two excites wave band as exciting light；Select 470 ± 10nm, 490 ± 10nm, 510 ± 10nm, 530 ± 10nm, 550
Six emission bands of ± 10nm and 585 ± 20nm are used as detection channels.Measured and calculating can obtain swashing for normalized C
Hair spectrumAnd emission spectraAnd normalized V PLEAnd emission spectra
(3) basisWithApposition obtain the excitation-emission spectrum basic vector (S of donorD),WithApposition obtain
The excitation-emission spectrum basic vector (S of acceptorA), andWithApposition obtain for acceptor be sensitized excitation-emission spectrum basic vector
(SS)：
(4) measurement donor-acceptor concatenation sample C32V, C17V and CTV excitation-emission spectrum (SDA[i]).With 436 ±
10nm exciting light excites each donor-acceptor concatenation sample respectively, respectively obtains transmission channel 470 ± 10nm, 490 ±
The fluorescence of each donor-acceptor concatenation sample is strong under 10nm, 510 ± 10nm, 530 ± 10nm, 550 ± 10nm and 585 ± 20nm
Degree；Excite each donor-acceptor concatenation sample respectively with 470 ± 10nm exciting light again, respectively obtain transmission channel 510 ±
The fluorescence intensity of each donor-acceptor concatenation sample under 10nm, 530 ± 10nm, 550 ± 10nm and 585 ± 20nm, so as to obtain
Excitation-emission spectrum (the S of each donor-acceptor concatenation sampleDA[i])。
(5) according to formula (2) by SDA[i]Linear separation is carried out according to three excitation-emission spectrum basic vectors, three weights are obtained
Factor WD[i]、WA[i]And WS[i]。
SDA[i]=WD[i]SD+WS[i]SS+WA[i]SA (2)
(6) with WA/WDFor independent variable, WS/WDFor dependent variable, the W of 3 donor-acceptor concatenation samples of linear fitA[i]/
WD[i], WS[i]/WD[i], the inverse of the slope of linear equation is KA/KD, the absolute value of the intercept of linear equation is QA/QD。
The general principle of the present invention is as follows：
(1) for one both contain free donor and free acceptor, and the FRET samples containing Donor Acceptor pair its
Excitation-emission spectrum is the linear combination of following four kinds of excitation-emission spectrum：The excitation-emission spectrum of free donor and acceptor knot
Synthesize to the excitation-emission spectrum of donor, the excitation-emission spectrum that is sensitized for acceptor and directly excite the excitation-emission light of acceptor
Spectrum.As shown in formula (3).
Wherein, E is the FRET efficiency of paired D-A in FRET samples；CdIt is free donor in FRET samples
Concentration；CdaIt is the concentration of paired D-A；It is the total concentration of acceptor in FRET samples；
(2) simplifying formula (3) can obtain：
Wherein,For the total concentration of donor in FRETAnd have：
(3) the normalized apparent FRET efficiency (E of acceptor density is pressedA) and by the normalized apparent FRET efficiency of donor concentrations
(ED) can be expressed as：
(4) simultaneous formula (5) and formula (6) can be obtained：
(5) it is n for acceptor number and donor number ratioA/nDDonor-acceptor concatenation sample, have EA×nA=ED×
nD, then formula (7) can be written as：
(6) 2 kinds or two or more acceptor number and donor number ratio (n are measuredA/nD) the identical and different confession of FRET efficiency
Body-acceptor concatenation sample can obtain multigroup WA/WD, WS/WD.With WA/WDFor independent variable, WS/WDIt is dependent variable to multigroup WA/WD,
WS/WDLinear fit is carried out, fitting obtains the slope and n of linear equationA/nDThe inverse of product be KA/KD, linear equation
The absolute value of intercept is QA/QD。
The present invention has the following advantages and effect relative to prior art：
The present invention is a kind of while measuring the fluorophor as donor and the fluorophor as acceptor in given survey
Measure the Q under system conditionA/QDAnd KA/KDMethod, with that measurement process is simple, time of measuring is short and measurement result is stable is excellent
Point.Therefore, the present invention is applied to different detecting systems, is quantitatively detected with important application value for living cells FRET,
The success rate of living cells FRET detections will be greatly enhanced, so as to promote FRET quantitative measurement technologies in cell biology
Using.
Brief description of the drawings
Fig. 1 is donor (SD) and acceptor (SA) and for acceptor sensitization (SS) excitation-emission spectrum basic vector；Wherein, (a) is left
Figure is the excitation spectrum image for singly turning C and V；White portion is the selected cell compartment (Cell) fluoresced, and gray area is institute
Select background area (BG)；(a) (a) left figure list turns the average intensities of the corresponding white portions of C and V and subtracts grey area according to right figure
What the average intensity in domain was obtained excites fluorescent intensity to compose；(b) left figure is the emission spectrum image for singly turning C and V；White portion is institute
The cell compartment (Cell) of fluorescence is published, gray area is selected background area (BG)；(b) (b) left figure list turns C according to right figure
The transmitting fluorescent intensity spectrum that the average intensity that the average intensity of white portion corresponding with V subtracts gray area is obtained；(c) left figure
It is normalized donorAnd acceptorPLE, (c) right figure is normalized donorAnd acceptor
Emission spectra；(d) it is donor (SD), acceptor (SA) and for acceptor sensitization (SS) excitation-emission spectrum basic vector.
Fig. 2 is the ratio W that three weight coefficients are measured using donor-acceptor concatenation sample C32VA[1]/WD[1]、WS[1]/
WD[1]；Wherein, (a) upper figure is the excitation-emission spectrum picture for expressing C32V in HepG2 cells；(a) in, figure below be respectively pair
The W answeredA[1]/WD[1]、WS[1]/WD[1]Pcolor and column diagram；(b) it is with WA/WDFor independent variable, WS/WDFor dependent variable Linear Quasi
Close C32V, C17V and CTV WA[i]/WD[i]、WS[i]/WD[i]The linear equation expression formula being worth to that is averaged of (i=1,2,3).Its
The inverse of the slope of middle linear equation is KA/KD, the absolute value of the intercept of linear equation is QA/QD。
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
1st, plasmid origin
Donor plasmid Cerulean (C), receptor plasmid are Venus (V) and FRET equal with reference to plasmid C32V, C17V and CTV
It is purchased from U.S. addgene plasmids storehouse.
2. wide field spectrum micro imaging system
Wide field fluorescence microscope originates from Japanese Olympus company, model IX73.Light source is Japanese Olympus
The mercury lamp of HGLGPS series.Object lens are that multiplication factor is the oil mirror that 40, numerical aperture is 1.3 (40 × 1.3NA), and one is equipped with four
The individual runner that excites for exciting piece, one (can install in each cube equipped with eight cube and excite piece, light splitting piece, sheet emitting each one
It is individual) runner, an electronic transmitting runner equipped with six sheet emittings, an external CCD camera.Excitation wavelength is by rotating
Runner is excited to be selected.
3rd, cell culture and plasmid transfection
HepG2 cells come from GuangZhou, China Ji'nan University, and the NBCS for adding 10% with DMEM culture mediums, which is placed on, to be contained
Have in 37 DEG C of incubator of 5% carbon dioxide and cultivate.It is cells trypsinised, go in Tissue Culture Dish, culture 24
After hour, when cell growth is to 70~90%, with in-vitro transfection reagent TurbofectTMCell is transferred to by plasmid is of short duration.
The specific steps of transfection：(1) DMEM that 40 μ L serum-frees are first added in the EP pipes of two sterilizings, each EP pipes is taken,
Then added into an EP pipe in 1~2 μ L transfection reagent, another EP pipes and add 1~2 μ L's (500~600ng/ μ L)
Plasmid, stands 5 minutes；After (2) 5 minutes, two EP pipes are mixed, 20 minutes, (3) 20 minutes are stood after gently blowing and beating 6~8 times
Afterwards, the DMEM of 420 μ L serum-free is added in the EP pipes just mixed, gently mixed；(4) cultivated with the DMEM of serum-free
Cell 2~3 times in base or PBS culture dish, mainly washes away that dead cell etc. is dirty, then the mixture in above-mentioned (3)
Move on in culture dish, culture dish is placed back in incubator 4~6 hours；After (5) 4~6 hours, transfection liquid, Ran Houyong are sucked
With cell 2~3 times in the DMEM culture mediums or PBS culture dish of serum-free, then toward adding containing newborn ox blood in culture dish
Clear DMEM culture mediums, culture can be used to experiment for 24~48 hours.
4. measure donor-acceptor concatenation sample
4.1 singly turn C samples, V samples and donor-acceptor concatenation sample CTV, C17V and C32V respectively, measure every kind of supply
The weight factor W that three excitation-emission spectrum are respectively accounted in body-acceptor concatenation sampleD[i], WA[i]And WS[i]。
Measure three excitation-emission spectrum basic vectors.The culture of single expression donor C and acceptor V HepG2 cells will be loaded with
Ware is placed in objective table, and 405 ± 10nm of selection, 436 ± 10nm, 470 ± 10nm and 480 ± 10nm tetra- excite wave band as exciting
Light, 470 ± 10nm, 490 ± 10nm, 510 ± 10nm, 530 ± 10nm, six emission bands of 550 ± 10nm and 585 ± 20nm are made
For detection channels, C and V PLE (Fig. 1 (a)) and C and V emission spectra (Fig. 1 (b)) are measured.The C's obtained after normalization
PLE (Fig. 1 (c) is left) and emission spectra (Fig. 1 (c) is right)；
WillWithSubstitute into the excitation-emission spectrum basic vector (S that formula (1) calculates donorD), acceptor
Excitation-emission spectrum basic vector (SD) and for the excitation-emission spectrum basic vector (S of acceptor sensitizationS), such as shown in Fig. 1 (d).
The weight factor that three excitation-emission spectrum is respectively accounted for is measured using donor-acceptor concatenation sample C32V, C17V and CTV
WD[i], WA[i]And WS[i].Wherein measure donor-acceptor concatenation sample C32V excitation-emission spectrum (SDA[1]) image (such as Fig. 2 (a)
Shown in upper figure), by SDA[1]Linear separation is carried out according to three excitation-emission spectrum basic vectors, three weight factor W are obtainedD[1],
WA[1]And WS[1].And obtain W by calculatingA[1]/WD[1]、Ws[1]/WD[1]Value.In Fig. 2 (a), down be respectively corresponding WA[1]/
WD[1]、Ws[1]/WD[1]Pcolor and column diagram.Statistics general 36 cells in 14 visuals field obtain WA[1]/WD[1]=0.699 ±
0.014、Ws[1]/WD[1]=0.737 ± 0.033；Same method measurement C17V, statistics general 32 cells in 13 visuals field are obtained
WA[2]/WD[2]=0.786 ± 0.027, Ws[2]/WD[2]=1.109 ± 0.040；CTV is measured, 4 visuals field are counted, it is general 14 thin
Born of the same parents obtain WA[3]/WD[3]=0.498 ± 0.002, Ws[3]/WD[3]=3.77 × 10-6±7.33×10-6.With WA/WDFor independent variable,
WS/WDFor dependent variable (i=1,2,3), linear fit WA[i]/WD[i]、WS[i]/WD[i].Obtained linear equation expression formula such as Fig. 2
(b) shown in, the inverse of the wherein slope of linear equation is KA/KD=1/3.850, the absolute value of the intercept of linear equation is QA/QD
=1.925.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (3)
1. one kind is separated based on excitation-emission spectrum and measures the ratio between Acceptor-Donor quantum yield and Acceptor-Donor delustring system simultaneously
The method of the ratio between number, it is characterised in that comprise the following steps：
(1) 2 kinds of measurement or the D-A string that two or more acceptor number is identical from donor number ratio and FRET efficiency is different
Join the excitation-emission spectrum S of sampleDA[i], and by SDA[i]Linear separation, which is carried out, according to three excitation-emission spectrum basic vectors obtains three
Individual weight factor WD[i], WA[i]And WS[i]；Wherein subscript i represents i-th kind of donor-acceptor concatenation sample, i >=2；
Three described excitation-emission spectrum basic vectors are respectively the excitation-emission spectrum basic vector S of donorD, acceptor excitation-emission light
Compose basic vector SA, for acceptor be sensitized excitation-emission spectrum basic vector SS；
Described WD[i]Weight, W shared by donor excitation-emission spectrumA[i]Weight, W shared by acceptor excitation-emission spectrumS[i]For
For weight shared by acceptor sensitization excitation-emission spectrum；
(2) W obtained according to step (1)D[i]、WA[i]And WS[i]Calculate WA[i]/WD[i]And WS[i]/WD[i]Value；
(3) with WA/WDFor independent variable, WS/WDFor dependent variable, the W obtained in linear fit step (2)A[i]/WD[i]、WS[i]/WD[i]；
The slope and n of linear equationA/nDThe inverse of product be KA/KD, the absolute value of the intercept of linear equation is QA/QD。
2. it is according to claim 1 separated based on excitation-emission spectrum measure simultaneously the ratio between Acceptor-Donor quantum yield with
The method of the ratio between Acceptor-Donor extinction coefficient, it is characterised in that：
Donor-acceptor concatenation sample described in step (1) is applied to the donor-acceptor concatenation sample of all Plasmid series.
3. it is according to claim 2 separated based on excitation-emission spectrum measure simultaneously the ratio between Acceptor-Donor quantum yield with
The method of the ratio between Acceptor-Donor extinction coefficient, it is characterised in that：
Acceptor number and donor number ratio are 0.5~3 in donor-acceptor concatenation sample described in step (1).
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| CN107271422B (en) | 2019-08-23 |
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Effective date of registration: 20211202 Address after: No.10, t0218 / F, Tianan Zhigu science and Technology Industrial Park, 18 Chuangxing Avenue, Qingyuan high tech Industrial Development Zone, Guangdong 511500 Patentee after: Normal University Rayleigh Optoelectronic Technology (Qingyuan) Co.,Ltd. Address before: 510631 No. 55, Zhongshan Avenue, Tianhe District, Guangdong, Guangzhou Patentee before: SOUTH CHINA NORMAL University |