CN107271422B - 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 - Google Patents
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 Download PDFInfo
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- G—PHYSICS
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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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The present invention discloses a kind of method for separating based on excitation-emission spectrum while measuring the ratio between Acceptor-Donor quantum yield with the ratio between extinction coefficient, belongs to FRET detection technique field.This method comprises the following steps: the excitation-emission spectrum S of 2 kinds of measurement or two or more receptor number is identical as donor number ratio and FRET efficiency is different donor-acceptor concatenation 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 suitable for different detection systems.
Description
Technical field
The invention belongs to fluorescence resonance energy transfer (FRET) detection technique fields, and in particular to one kind is based on excitation-emission
Spectrum separates while measuring the ratio between Acceptor-Donor quantum yield (QA/QD) with the ratio between Acceptor-Donor extinction coefficient (KA/KD) side
Method.
Background technique
FRET microscopy based on fluorescin (FPs) has become studies the important of molecular regulation mechanism in living cells
Tool.Obtaining independent of the quantitative FRET signal of detection system and FPs expression is that science is carried out between different experiments room
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 measuring technique (ExEm-spFRET) for composing separation can overcome receptor excitation 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
Essential two Important Parameters of FRET detection technique.KA/KDSpectral property, measuring system excitation channel with excitation light source
Transfer function and donor it is related with the absorption spectrum of receptor.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. by measure Plasmid series sample exist
Rate of induced polarization when dual wavelength excites obtains KA/KD, this method requires system setting in all FRET measurement process to remain unchanged.
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. mention
Sp-ECR method out measures KA/KDWhen need by Plasmid series sample known to a FRET efficiency.For QA/QDValue,
Most of FRET correlative studys are obtained by the quantum yield value for the fluorophor reported in citation.But actually
QA/QDIt is not only related with the optical property of donor, acceptor fluorophore, also with environment locating for fluorophor and measuring system
The spectral response performance of transmission channel is related.So measuring Q simultaneously and accurately in given measuring systemA/QDAnd KA/KD's
Value is the precondition of a variety of quantitative FRET detections.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide one kind to be based on excitation-emission spectrum
It separates while measuring the ratio between Acceptor-Donor quantum yield (QA/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 receptor number is identical as donor number ratio and FRET efficiency is different
Sample measures Q simultaneouslyA/QDAnd KA/KD。
The purpose of the invention is achieved by the following technical solution:
Single-turn donor sample, single-turn receptor sample and 2 kinds or two or more receptor number and donor number ratio are obtained respectively
It is worth donor-acceptor concatenation samples identical and that FRET efficiency is different.It is sent out by the excitation of linear separation donor-acceptor concatenation sample
Q under the conditions of given measuring system can quickly be measured by penetrating spectrumA/QDAnd KA/KDValue, and without measuring donor-receptor string
Join the FRET efficiency of sample.
One kind is separated based on excitation-emission spectrum while measuring the ratio between Acceptor-Donor quantum yield (QA/QD) and receptor-confession
The ratio between body extinction coefficient (KA/KD) method, comprising the following steps:
(1) 2 kinds of measurement or two or more receptor number and donor number ratio (nA/nD) identical and confession that FRET efficiency is different
Body-acceptor concatenation sample excitation-emission spectrum (SDA[i]), and by SDA[i]It is 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 indicates 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, 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 receptor sensitization excitation-emission spectrum basic vector SS；
W described in step (1)D[i]For weight shared by donor excitation-emission spectrum, WA[i]For receptor excitation-emission spectrum institute
Account for weight, WS[i]To be sensitized weight shared by excitation-emission spectrum for receptor；
Donor-acceptor concatenation sample described in step (1) is suitable for the donor-acceptor concatenation sample of all Plasmid series
This, wherein receptor number and donor number ratio (nA/nD) it is preferably 0.5~3；Most preferably 1:1.
Specifically includes the following steps:
(1) single-turn donor sample and single-turn receptor sample are obtained, the excitation-emission spectrum basic vector S of donor is measuredD, receptor
Excitation-emission spectrum basic vector SA, and for the excitation-emission spectrum basic vector S of receptor sensitizationS；
(2) 2 kinds of acquisition or two or more receptor number and donor number ratio (nA/nD) identical and confession that FRET efficiency is different
Body-acceptor concatenation sample measures the excitation-emission spectrum (S of donor-acceptor concatenation sampleDA[i]), and by SDA[i]Swash according to three
It sends out emission spectrum basic vector and carries 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 relationship:
In order to preferably illustrate the present invention, below with a measurement QA/QDAnd KA/KDExample be illustrated:
Given donor-acceptor concatenation sample (nA/nD=1): donor is Cerulean (abbreviation C), and receptor is Venus (letter
Claim V).
Donor-acceptor concatenation sample C32V: C and V is linked into the donor-receptor formed by the catenation sequence of 32 amino acid
Series connection sample.
Donor-acceptor concatenation sample C17V: C and V is linked into the donor-receptor formed by the catenation sequence of 17 amino acid
Series connection sample.
Donor-acceptor concatenation sample CTV: C and V is linked into the donor-receptor formed by the catenation sequence of 229 amino acid
Series connection sample.
Specific measurement process is as follows:
(1) individually transfect and express in the liver cancer cells of people (HepG2 cell) donor C and receptor V and donor-by
Body series connection sample C32V, C17V and CTV；
(2) excitation spectrum and emission spectra of wide field fluorescence microscope and camera measurement C and V are used.Select 436 ± 10nm and 470
Two excitation wave bands of ± 10nm are as exciting light；Select 470 ± 10nm, 490 ± 10nm, 510 ± 10nm, 530 ± 10nm, 550
Six emission bands of ± 10nm and 585 ± 20nm are as detection channels.By measuring and calculating swashing for available normalized C
Hair spectrumAnd emission spectraAnd the excitation spectrum of normalized VAnd emission spectra
(3) basisWithApposition obtain the excitation-emission spectrum basic vector (S of donorD),WithApposition obtain
The excitation-emission spectrum basic vector (S of receptorA), andWithApposition obtain for receptor be sensitized excitation-emission spectrum basic vector
(SS):
(4) the excitation-emission spectrum (S of donor-acceptor concatenation sample C32V, C17V and CTV are measuredDA[i]).With 436 ±
The exciting light of 10nm excites each donor-acceptor concatenation sample respectively, respectively obtains 470 ± 10nm of transmission channel, 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 the exciting light of 470 ± 10nm again, respectively obtain transmission channel 510 ±
The fluorescence intensity of each donor-acceptor concatenation sample under 10nm, 530 ± 10nm, 550 ± 10nm and 585 ± 20nm, 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, obtains three weights
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。
Basic principle of the invention is as follows:
(1) for one not only contain free donor and free receptor, but also the FRET sample 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 receptor knot
The excitation-emission light of the excitation-emission spectrum of the donor of synthesis pair, the excitation-emission spectrum and directly excitation receptor that are sensitized for receptor
Spectrum.As shown in formula (3).
Wherein, E is the FRET efficiency of donor-receptor pairs of in FRET sample；CdIt is free donor in FRET sample
Concentration；CdaIt is the concentration of pairs of donor-receptor；It is the total concentration of receptor in FRET sample；
(2) simplified 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 indicate are as follows:
(4) simultaneous formula (5) and formula (6) can obtain:
It (5) is n for receptor number and donor number ratioA/nDDonor-acceptor concatenation sample, have EA×nA=ED×
nD, then formula (7) can be written as:
(6) 2 kinds of measurement or two or more receptor number and donor number ratio (nA/nD) identical and confession that FRET efficiency is different
The available multiple groups W of body-acceptor concatenation sampleA/WD, WS/WD.With WA/WDFor independent variable, WS/WDIt is dependent variable to multiple groups 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 compared with the existing technology, have following advantages and effects
The present invention is a kind of while measuring the fluorophor as donor and the fluorophor as receptor 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 suitable for different detection systems, has important application value for living cells FRET quantitative detection,
The success rate that living cells FRET detection will be greatlyd improve, to push FRET quantitative measurement technology in cell biology
Using.
Detailed description of the invention
Fig. 1 is donor (SD) and receptor (SA) and for receptor sensitization (SS) excitation-emission spectrum basic vector；Wherein, (a) is left
Figure is the excitation spectrum image of single-turn C and V；White area is the selected cell compartment (Cell) to fluoresce, and gray area is institute
Select background area (BG)；(a) average intensity of the corresponding white area of (a) left figure single-turn C and V subtracts grey area according to right figure
The excitation fluorescent intensity spectrum that the average intensity in domain obtains；(b) left figure is the emission spectrum image of single-turn C and V；White area is institute
The cell compartment (Cell) of fluorescence is published, gray area is selected background area (BG)；(b) (b) left figure single-turn C according to right figure
The transmitting fluorescent intensity spectrum that the average intensity that the average intensity of white area corresponding with V subtracts gray area obtains；(c) left figure
It is normalized donorAnd receptorExcitation spectrum, (c) right figure is normalized donorAnd receptorEmission spectra；It (d) is donor (SD), receptor (SA) and for receptor 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 cell；(a) in, the following figure be pair respectively
The W answeredA[1]/WD[1]、WS[1]/WD[1]Pcolor and column diagram；It (b) is with WA/WDFor independent variable, WS/WDFor dependent variable Linear Quasi
Close the W of C32V, C17V and CTVA[i]/WD[i]、WS[i]/WD[i]The linear equation expression formula that the average value of (i=1,2,3) obtains.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。
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
1, plasmid origin
Donor plasmid Cerulean (C), receptor plasmid are Venus (V) and FRET is equal referring to plasmid C32V, C17V and CTV
It is purchased from U.S. addgene plasmid library.
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 the oil mirrors that amplification factor is 40, numerical aperture is 1.3 (40 × 1.3NA), and one is equipped with four
The excitation runner of a excitation piece, one equipped with eight cube (mountable excitation piece, light splitting piece, sheet emitting each one in each cube
It is a) runner, one dress there are six sheet emitting electronic transmitting runner, an external CCD camera.Excitation wavelength passes through rotation
Excitation runner is selected.
3, cell culture and plasmid transfection
HepG2 cell comes from GuangZhou, China Ji'nan University, is placed on and is contained with the newborn bovine serum that DMEM culture medium is added 10%
It is cultivated in 37 DEG C of incubator for having 5% carbon dioxide.It is cells trypsinised, it goes in Tissue Culture Dish, culture 24
After hour, when cell grows to 70~90%, with in-vitro transfection reagent TurbofectTMCell is transferred to by plasmid is of short duration.
The specific steps of transfection: (1) taking the EP pipe of two sterilizings, the DMEM of 40 μ L serum-frees be first added in each EP pipe,
Then the transfection reagent of 1~2 μ L is added into an EP pipe, 1~2 μ L (500~600ng/ μ L) is added in another EP pipe
Plasmid stands 5 minutes；(2) after five minutes, two EP pipes are mixed, stands 20 minutes, (3) 20 minutes after gently blowing and beating 6~8 times
Afterwards, the DMEM of the serum-free of 420 μ L is added in the EP pipe just mixed, is mixed gently；(4) it is cultivated with the DMEM of serum-free
Cell 2~3 times in base or PBS cleaning culture dish, mainly wash away that dead cell etc. is dirty, then the mixture in above-mentioned (3)
It moves on in culture dish, culture dish is placed back in incubator 4~6 hours；After (5) 4~6 hours, transfection liquid is sucked, is then used
Cell 2~3 times in culture dish are cleaned with the DMEM culture medium or PBS of serum-free, then are added into culture dish containing newborn ox blood
Clear DMEM culture medium, culture can be used to test for 24~48 hours.
4. measuring donor-acceptor concatenation sample
4.1 difference single-turn C samples, V sample and donor-acceptor concatenation sample CTV, C17V and C32V, measure every kind of confession
The weight factor W that three excitation-emission spectrum respectively account in body-acceptor concatenation sampleD[i], WA[i]And WS[i]。
Measure three excitation-emission spectrum basic vectors.The culture of the HepG2 cell of single expression donor C and receptor V will be loaded with
Ware is placed in objective table, selects tetra- 405 ± 10nm, 436 ± 10nm, 470 ± 10nm and 480 ± 10nm excitation wave bands as excitation
Light, 470 ± 10nm, 490 ± 10nm, 510 ± 10nm, 530 ± 10nm, six emission bands of 550 ± 10nm and 585 ± 20nm are made
For detection channels, the excitation spectrum (Fig. 1 (a)) of C and V and the emission spectra (Fig. 1 (b)) of C and V are measured.The C's obtained after normalization
Excitation spectrum (Fig. 1 (c) is left) and emission spectra (Fig. 1 (c) is right)；
It willWithSubstitute into the excitation-emission spectrum basic vector (S that formula (1) calculates donorD), receptor
Excitation-emission spectrum basic vector (SD) and for receptor sensitization excitation-emission spectrum basic vector (SS), as shown in Fig. 1 (d).
The weight factor that three excitation-emission spectrum respectively accounts for is measured using donor-acceptor concatenation sample C32V, C17V and CTV
WD[i], WA[i]And WS[i].Wherein measure the excitation-emission spectrum (S of donor-acceptor concatenation sample C32VDA[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, obtains three weight factor WD[1],
WA[1]And WS[1].And W is obtained by calculationA[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.It counts general 36 cells in 14 visuals field and obtains WA[1]/WD[1]=0.699 ±
0.014、Ws[1]/WD[1]=0.737 ± 0.033；Same method measures C17V, counts general 32 cells in 13 visuals field and obtains
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, wherein inverse, that is, K of the slope of linear equationA/KD=1/3.850, absolute value, that is, Q of the intercept of linear equationA/QD
=1.925.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (3)
1. one kind is separated based on excitation-emission spectrum while measuring the ratio between Acceptor-Donor quantum yield and Acceptor-Donor delustring system
The method of the ratio between number, it is characterised in that the following steps are included:
(1) 2 kinds of measurement or the donor-receptor string that two or more receptor number is identical as 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
A weight factor WD[i], WA[i]And WS[i]；Wherein subscript i indicates i-th kind of donor-acceptor concatenation sample, i >=2；
Three excitation-emission spectrum basic vectors are respectively the excitation-emission spectrum basic vector S of donorD, receptor excitation-emission light
Compose basic vector SA, for receptor sensitization excitation-emission spectrum basic vector SS；
The WD[i]For weight shared by donor excitation-emission spectrum, WA[i]For weight shared by receptor excitation-emission spectrum, WS[i]For
For weight shared by receptor 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, 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；
The nA/nDIndicate receptor number and donor number ratio；The KA/KDIndicate the ratio between Acceptor-Donor extinction coefficient；
The QA/QDIndicate the ratio between Acceptor-Donor quantum yield.
2. it is according to claim 1 based on excitation-emission spectrum separate and meanwhile measure 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 suitable for the donor-acceptor concatenation sample of all Plasmid series.
3. it is according to claim 2 based on excitation-emission spectrum separate and meanwhile measure the ratio between Acceptor-Donor quantum yield with
The method of the ratio between Acceptor-Donor extinction coefficient, it is characterised in that:
Receptor number and donor number ratio are 0.5~3 in donor-acceptor concatenation sample described in step (1).
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