CN101551332A - Method for quantitatively measuring transferring efficiency of fluorescence resonance energy by utilizing fluorescence spectrum fitting - Google Patents
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Abstract
The present invention relates to method for measuring transferring efficiency of fluorescence resonance energy, particular providing a method for quantitatively measuring transferring efficiency of fluorescence resonance energy by utilizing fluorescence spectrum fitting. Steps of the method includes first measuring and obtaining fluorescence-emission spectrum of donor-receptor inside cells; and then utilizing fluorescence-emission spectrum of normalized donor and receptor to obtain fitting fluorescence spectrum of donor-receptor; at last calculating transferring efficiency of fluorescence resonance energy at the closest of experiment optical spectrum and fitting optical spectrum. Through method of the invention, being able to resolve problems of difficult quantitatively measure of FRET efficiency caused by spectroscopic excitation crossfire and transmission crossfire effectively, consequently can monitoring activate degree of protein and reciprocity among proteins in live cells more accurately.
Description
Technical Field
The invention relates to a method for measuring fluorescence resonance energy transfer efficiency, in particular to a method for quantitatively measuring fluorescence resonance energy transfer efficiency by utilizing fluorescence spectrum fitting.
Background
Various technologies based on fluorescence have become important technologies for detecting molecular regulatory mechanisms of cell signal transduction, apoptosis and proliferation in real time in living cells. The fluorescence spectrum technology is widely applied to the analysis of chemical compositions, protein components and structures, and also widely applied to the analysis of tumor characteristics, traditional Chinese medicine components and traditional Chinese medicine action mechanisms. Fluorescence Resonance Energy Transfer (FRET) is a technique for quantitatively measuring the distance between two different luminescent groups. FRET is a process of transferring the energy of a donor excited state to an acceptor excited state through intermolecular electric dipole interaction, which is a non-radiative energy transfer. When FRET occurs, donor fluorescence decreases and acceptor fluorescence increases, and if the increased fluorescence intensity of the acceptor and the decreased fluorescence intensity of the donor can be accurately measured, the FRET efficiency can be accurately measured. However, since emission spectra of the donor and the acceptor are generally crossed, and the shortest wavelength of the laser confocal scanning microscope widely used in biological research at present is 458nm, the donor and the acceptor are excited simultaneously, so that it is difficult to eliminate excitation crosstalk and emission crosstalk in actual measurement, and therefore, measured data must be processed to eliminate the influence caused by various kinds of crosstalk. There are several methods for calculating FRET efficiency, Gordon [ Gordon G W, Berry G, Liang X H, Levine B, Herman B.quantitative fluorescence resonance transduction using fluorescence microscopy. Biophysical journal, 1998, 74: 2702-2713 ] proposes a method for quantitatively calculating FRET efficiency that can correct emission crosstalk, which is very complicated by using three different sets of filters to measure the fluorescence intensity of separate donor and acceptor and donor-acceptor pairs in the three sets of filters, respectively. In addition, this method is not suitable for immobilized donor-acceptor pairs because it is difficult to obtain separate donor and acceptor pairs. Aging [ Chen T S, ZengS Q, Luo Q M.fixing of fluoro restriction energy transfer efficiency means of emission spectra of doror-receptor pair, Acta Photonic Sinica, 2001, 30: 300-303 ] a method for quantitatively measuring FRET efficiency is proposed, which quantitatively obtains FRET efficiency by fitting using emission spectrum information of a donor-acceptor pair when a donor is selectively excited. However, the currently widely used laser confocal scanning microscope in the biological field is generally configured with an argon ion laser, the shortest excitation wavelength of which is 458nm, and the excitation light is difficult to selectively excite the donor, so the above method is not suitable for the case of excitation crosstalk.
Disclosure of Invention
Aiming at the problems of excitation crosstalk and emission crosstalk existing in the existing FRET efficiency measurement method, the invention aims to provide a novel method for quantitatively measuring the FRET efficiency by fitting fluorescence spectra of donor-acceptor pairs.
The invention is realized by the following technical scheme: a method for quantitatively measuring fluorescence resonance energy transfer efficiency by utilizing fluorescence spectrum fitting comprises the following specific steps:
a method for quantitatively measuring fluorescence resonance energy transfer efficiency by utilizing fluorescence spectrum fitting comprises the following specific steps:
(1) measuring fluorescence emission spectra of living cells before and after donor-acceptor pair transfection to obtain fluorescence emission spectra SP of pure donor-acceptor pairmeasure(λ)；
Measuring the excitation wavelength of 438 +/-20 nm when measuring the fluorescence emission spectrum, and scanning the emission spectrum in the range of 470-580 nm;
(2) respectively measuring fluorescence emission spectra of the donor and the acceptor, and normalizing to obtain standard emission spectrum SP of the acceptora(lambda) and donor standard emission Spectrum SPd(lambda), the theoretical emission spectrum SP is obtained by using the following formulanorm(λ)：
SPnorm(λ)＝SPnew(λ)/max(SPnew(λ))，
Wherein,
SPnew(λ)＝xφASPa(λ)+(1-x)φD[(1-E)*b*SPd(λ)+EφA*SPa(λ)/φD]，
wherein,
e is the energy resonance transfer efficiency, x is the excitation crosstalk, phiDIs the quantum yield of the donor (for ECFP φ)D＝0.40)，φAIs the quantum yield of the acceptor, (for Venus phi)A＝0.57)，
(3) Then calculate different E and x, SPnorm(lambda) and SPmeasureAnd (lambda) traversing E and x from 0 to 1 according to the standard deviation between the E and the x, fixing the value step length, and obtaining the fluorescence energy resonance transfer effect E and the excitation crosstalk x when the standard deviation is minimum.
To better implement the invention:
SACT3 was used for the donor-acceptor pair described above. The SACT3 plasmid constructed based on FRET principle consists of a cyan fluorescent protein (ECFP) donor, a Yellow Fluorescent Protein (YFP) mutant (Venus) acceptor and a segment of sequence containing caspase-3 cleavage substrate DEVD connected between the cyan fluorescent protein donor and the Yellow Fluorescent Protein (YFP) acceptor, and is mainly used for detecting activation of caspase-3.
In the step (1), the step length of scanning the spectrum is 2nm, and each point is continuously measured for 25 times and averaged.
The basic principle of the invention is as follows:
for confocal laser microscopy, the shortest excitation wavelength is typically 458nm, which results in simultaneous excitation of the donor and acceptor, whereas we typically need to selectively excite the donor. In the present invention, we used normalized fluorescence emission spectra of donors and acceptors to analyze energy resonance transfer efficiency quantitatively by fitting fluorescence spectra obtained from measurements.
First, let us assume the total excitation intensity as I0And the intensity of light used to excite the receptors is xI0Thus the fluorescence intensity obtained by direct excitation of the acceptor is Ia-d：
Ia-d＝xI0φA (1)
Here phiAIs the quantum yield of the acceptor, (for Venus phi)A0.57). While directly exciting the fluorescence intensity I generated by the donord+a：
Id+a＝(1-E)Id-a+φAEId-a/φD (2)
Here Id-aIs the intensity of fluorescence produced by exciting the donor in the absence of the acceptor, E is the efficiency of the energy resonance transfer, phiDIs the quantum yield of the donor (for ECFP φ)D0.40). Because of Id-a＝(1-x)I0φDSubstituting it into (2), we get:
Id+a＝(1-x)I0φD[(1-E)+φAE/φD] (3)
thus, in the presence of excitation crosstalk, the total fluorescence intensity is:
I＝Ia-d+Id+a
＝Ia-d+(1-x)I0φD[(1-E)+φAE/φD] (4)
by the formula (1), we obtain I0＝Ia-d(x φ A), substituting for (5):
we set the normalized donor and acceptor standard spectra to SPd(lambda) and SPa(lambda). And the wavelength interval of the standard spectrum is 2 nm. The fluorescence emission spectrum in the presence of excitation crosstalk obtained by (5) is:
here, the
Since the integration ranges on both sides of equation (6) are the same, we can assume that the following equation holds:
since only the normalized emission spectrum needs to be considered in our invention, x φ is multiplied simultaneously on both sides of equation (6)AWithout changing the shape of the spectrum SP (λ). We use SPnew(λ) instead of SP (λ), hence SPnew(λ) can be expressed as:
SPnew(λ)＝xφASPa(λ)+(1-x)φD[(1-E)*b*SPd(λ)+EφA*SPa(λ)/φD](8)
we pass max (SP)new(λ)) to normalize equation (7), where max (SP)new(lambda)) represents SPnewThe maximum value of (λ), and thus the normalized emission spectrum, can be expressed as SPnorm(λ)：
SPnorm(λ)＝SPnew(λ)/max(SPnew(λ)) (9)
Then we calculate SP at different E and xnorm(lambda) and experimentally obtained fluorescence emission Spectrum SPmeasure(λ) standard deviation (E and x are both from 0 to 1). The fluorescence energy resonance transfer effect E and the excitation crosstalk x can be obtained by least square fitting. And E and x are traversed from 0 to 1 during fitting, the value step length is fixed, and when the standard deviation is minimum, the corresponding values of E and x are the fluorescence energy resonance transfer effect E and the excitation crosstalk ratio x.
Compared with the prior art, the invention has the following beneficial effects:
the invention can effectively solve the problem that the FRET efficiency is difficult to be quantitatively measured due to spectrum excitation crosstalk and emission crosstalk. By measuring the fluorescence spectrum of the donor-acceptor in real time, the FRET efficiency between the donor-acceptor pair can be obtained quantitatively by using the method, so that the activation degree of the protein can be monitored. In addition, because FRET efficiency can also reflect the binding degree between proteins, the interaction between proteins in living cells can be quantitatively monitored by using the method.
Drawings
FIG. 1 is a spectrum of SCAT3 in living cells that were not treated with staurosporine.
FIG. 2 is a spectrum of SCAT3 in living cells after 12 hours of staurosporine treatment.
FIG. 3 is a spectrum of viable cells that were not transfected with SCAT 3.
FIG. 4 is a spectrum of SCAT3 after subtraction of background spectrum in live cells that were not treated with staurosporine.
FIG. 5 is a spectrum of SCAT3 in live cells after 12 hours of staurosporine treatment minus the background spectrum.
FIG. 6 is a normalized fluorescence spectrum of the donor.
FIG. 7 is a normalized fluorescence spectrum of the acceptor.
FIG. 8 is a graph showing the spectrum of SCAT3 in live cells not treated with staurosporine minus the background spectrum and normalized SPmeasure(λ) the FRET efficiency was 25.4% as a result of fitting to the formula (8). , + ++ represent experimental data, and- -represents fitting data.
FIG. 9 is a graph showing the spectrum of SCAT3 in live cells treated with staurosporine for 12 hours, minus the background spectrum, and normalized SPmeasure(λ) the FRET efficiency was 4.5% as a result of fitting to the formula (8). , + ++ represent experimental data, and- -represents fitting data.
Detailed Description
The present invention is further described below with reference to the drawings and examples, but the embodiments of the present invention are not limited thereto.
1. Plasmid origin:
plasmid SCAT3 the plasmid was purchased from merck, germany.
2. Cell culture
Human Lung adenocarcinoma cells (ASTC-a-1) were grown in DMEM medium containing 10% newborn bovine serum, 50units/ml penicillin and 50g/ml streptomycin, and after 70-85% confluence, they were trypsinized and passaged to 1X 104The cells were plated at intervals of 500. mu.l/well in cell culture dishes, and after plating, they were placed in an incubator (37 ℃ C., 5% CO2) for further culture. Transforming SCAT3 plasmid into ASTC-a-1 cells through liposome, and screening cells transfected with SCAT3 by G418 to obtain ASTC-a-1 cells stably expressing SCAT3 [ Wu Y X, Xing D, Chen W R.Single cell FRET imagining for determination of pathway of tumor cell apoptosis induced by apoptosis-PDT.cell Cycle, 2006, 5: 729-734; wu Y X, Xing D, Luo S M, Tang Y H, Chen Q.Detection of caspase-3 activation in single cells by fluorescence emission energy transfer luminescence therapy cancer Letter, 2006, 235: 239-247.]。
3. Measurement of fluorescence resonance energy transfer efficiency.
G418 is used for screening the cells transfected with SCAT3 to obtain ASTC-a-1 cells stably expressing SCAT 3. ASTC-a-1 cells which express SCAT3 and are stably transfected are inoculated into a 96-well plate to be cultured for 24 hours, and then 100L of staurosporine with the concentration of 1mol/L is added into each well after culture solution is replaced. The fluorescence emission spectra of the donor-acceptor pairs in living cells were then measured using an automated microwell detector (infinite M200, Tecan, Austria). The excitation wavelength is 438 +/-20 nm, the emission spectrum is scanned in the range of 470-580nm, the scanning spectrum step size is 2nm, and each point is continuously measured for 25 times and averaged. The measured spectra are shown in fig. 1 and 2: FIG. 1 is a spectrum of SCAT3 in living cells that were not treated with staurosporine. FIG. 2 is a spectrum of SCAT3 in living cells after 12 hours of staurosporine treatment.
(1) The corresponding emission spectrum of the cells not transfected with SCAT3 was measured as background spectrum. The excitation wavelength is 438 +/-20 nm, the emission spectrum is scanned in the range of 470-580nm, the scanning spectrum step size is 2nm, and each point is continuously measured for 25 times and averaged. The measured background spectrum is shown in fig. 3.
(2) Subtracting the background spectrum from the fluorescence spectrum of the cells stably transfected and expressing the donor-acceptor pair and normalizing to obtain the fluorescence spectrum SP of the pure donor-acceptor pairmeasure(λ) as shown in fig. 4 and 5.
(3) Measuring the emission spectra of the donor and the acceptor, and normalizing to obtain an acceptor standard emission spectrum SPa(lambda) and donor standard emission Spectrum SPd(λ), normalized emission spectra of the donor and acceptor are shown in FIGS. 6 and 7 below.
The theoretical emission spectrum SP is obtained by using the following formulanorm(λ)：
SPnorm(λ)＝SPnew(λ)/max(SPnew(λ))，
Wherein,
SPnew(λ)＝xφASPa(λ)+(1-x)φD[(1-E)*b*SPd(λ)+EφA*SPa(λ)/φD]，
where E is the energy resonance transfer efficiency and x is the excitation crosstalk, phiDIs the quantum yield of the donor (for ECFP φ)D＝0.40)，φAIs the quantum yield of the acceptor, (for Venus phi)A＝0.57)
SPnew(λ)＝0.57x*SPa(λ)+0.4(1-x)[0.6(1-E)*SPd(λ)+1.425E*SPa(λ)]
SPnorm(λ)＝SPnew(λ)/max(SPnew(λ))
(4) Then calculate SP at different E and xnorm(lambda) and experimentally obtained fluorescence emission Spectrum SPmeasure(λ) standard deviation (E and x are both from 0 to 1). And E and x are traversed from 0 to 1 during fitting, the value step is 0.01, and the fluorescence energy resonance transfer effect E and the excitation crosstalk x are obtained when the standard deviation is minimum.
Based on the above calculations, the measured spectrum of SCAT3 in live cells that were not treated with STS and treated with STS for 12 hours subtracted with the background spectrum and normalized the resulting SPmeasure(lambda) and SPnormThe results of the (λ) fitting are shown in fig. 8 and 9.
As seen from this example, the FRET efficiency E of SCAT3 was smaller in cells after 12 hours of staurosporine treatment, indicating that intracellular caspase3 was activated after staurosporine treatment, resulting in SCAT3 being cleaved, decreasing the FRET efficiency E of SCAT 3. When caspase3 is activated, cells can enter apoptosis irreversibly, so that apoptosis of cells can be detected by the invention.
In addition, the results of this example can be confirmed by fluorescence lifetime imaging microscopy. The fluorescence lifetime imaging experiments were performed on live cells stably expressing SCAT3 treated with staurosporine and 12 hours after the untreated and treated cells, and the FRET efficiencies E obtained were 24.4% and 3.7%, respectively, which are substantially identical to the results obtained with the present invention.
Claims (3)
1. A method for quantitatively measuring fluorescence resonance energy transfer efficiency by utilizing fluorescence spectrum fitting comprises the following specific steps:
(1) measuring fluorescence emission spectra of living cells before and after donor-acceptor pair transfection to obtain fluorescence emission spectra SP of pure donor-acceptor pairmeasure(λ)；
Measuring the excitation wavelength of 438 +/-20 nm when measuring the fluorescence emission spectrum, and scanning the emission spectrum in the range of 470-580 nm;
(2) measuring fluorescence emission spectra of donor and acceptor respectively, and normalizing to obtain acceptor standard emissionEmission spectrum SPa(lambda) and donor standard emission Spectrum SPd(lambda), the theoretical emission spectrum SP is obtained by using the following formulanorm(λ)：
SPnorm(λ)＝SPnew(λ)/max(SPnew(λ))，
Wherein,
SPnew(λ)＝xφASPa(λ)+(1-x)φD[(1-E)*b*SPd(λ)+EφA*SPa(λ)/φD]，
wherein,
e is the energy resonance transfer efficiency, x is the excitation crosstalk, phiDIs the quantum yield of the donor, phiAIs the quantum yield of the acceptor and,
(3) then calculate different E and x, SPnorm(lambda) and SPmeasureAnd (lambda) traversing E and x from 0 to 1 according to the standard deviation between the E and the x, fixing the value step length, and obtaining the fluorescence energy resonance transfer effect E and the excitation crosstalk x when the standard deviation is minimum.
2. The method for quantitatively measuring fluorescence resonance energy transfer efficiency using fluorescence spectrum fitting according to claim 1, wherein: the donor-acceptor pair was SACT3, which included cyan fluorescent protein ECFP as the donor and a mutant Venus of yellow fluorescent protein YFP as the acceptor.
3. The method for quantitatively measuring fluorescence resonance energy transfer efficiency using fluorescence spectrum fitting according to claim 1, wherein: in the step (1), the step length during spectrum scanning is 2nm, and each point is continuously measured for 25 times and averaged.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636465A (en) * | 2011-10-26 | 2012-08-15 | 华南师范大学 | FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation |
| CN106442455A (en) * | 2016-11-23 | 2017-02-22 | 华南师范大学 | Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection |
| CN107924164A (en) * | 2015-06-22 | 2018-04-17 | 加利福尼亚大学董事会 | The quantitative measure of the interaction based on FRET |
| CN118050337A (en) * | 2024-01-31 | 2024-05-17 | 中国海洋大学 | Time-resolved fluorescence resonance energy transfer-based protein-small molecule ligand interaction analysis method |
-
2009
- 2009-05-14 CN CNA2009100394712A patent/CN101551332A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636465A (en) * | 2011-10-26 | 2012-08-15 | 华南师范大学 | FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation |
| CN107924164A (en) * | 2015-06-22 | 2018-04-17 | 加利福尼亚大学董事会 | The quantitative measure of the interaction based on FRET |
| CN106442455A (en) * | 2016-11-23 | 2017-02-22 | 华南师范大学 | Method for quickly measuring fluorescence resonance energy transfer (FRET) efficiency based on simultaneous dual-channel fluorescence intensity detection |
| CN106442455B (en) * | 2016-11-23 | 2019-01-08 | 华南师范大学 | A kind of method for fast measuring of the transferring efficiency of fluorescence resonance energy detected simultaneously based on binary channels fluorescence intensity |
| CN118050337A (en) * | 2024-01-31 | 2024-05-17 | 中国海洋大学 | Time-resolved fluorescence resonance energy transfer-based protein-small molecule ligand interaction analysis method |
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