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1.IntroductionFluorescence resonance energy transfer (FRET) has become an important tool for the analysis of intracellular biological processes1 and biological sensor applications.2 Recent publications have shown that a multiple acceptors-based FRET technique has the potential to characterize the functions of multiprotein complexes in living cells and the spatial conformation of macromolecules on a scale larger than 10 nm.3,4 Multiple acceptors interacting with the same donor increase the probability of FRET even without a change in molecular distance and thus lead to high transfer efficiency.5 For example, FRET constructs with one donor and 10 acceptors can be used to determine distances as long as 20 nm.6 It was found that the amount of energy transfer observed in constructs with multiple acceptors (, “” denotes donor molecule and “” denotes acceptor molecule, represents the number of acceptors in the FRET construct) is significantly greater than the FRET efficiency predicated from the sum of the individual donor to acceptor transfer rates, indicating that either an additional energy transfer pathway exists when multiple acceptors are present or the theoretical assumption on which the kinetic model prediction is based is incorrect.7 Although FRET microscopy has become an important tool for monitoring intracellular biological processes, the contamination of spectral crosstalks, including donor emission crosstalk and acceptor excitation crosstalk, restricts the live cell applications of FRET.1,8 FRET quantification is very important for the quantitative comparison between different laboratories and/or cells with different expression levels of fluorescent proteins (FPs).8–11 Fluorescence lifetime imaging microscopy (FLIM), acceptor photobleaching (PbFRET), and acceptor-sensitized emission (SE-FRET) methods have been developed for FRET quantification.8 FLIM and PbFRET methods cannot provide the information about acceptor-to-donor concentration ratio (), and the long measurement time and irreversible destruction of photobleaching make the two methods incompatible with time-lapse live cell applications.8,12–14 SE-FRET method, the most widely used approach for dynamic live cell FRET applications,8 includes 3-cube-based methods such as E-FRET and spectral unmixing method (spFRET).9–11,15–19 The nondestructive nature (in contrast to the popular PbFRET technique) and the sensitivity even at high acquisition speed (in contrast to the fluorescence lifetime imaging) make E-FRET the method of choice for measurements over extended periods of time.8 spFRET is based on the axiom that the net fluorescence spectrum is defined by the linear superposition of the spectra for each fluorophore in the sample.19 Lux-FRET, a representative spFRET method developed by Wlodarczyk et al.,17 can resolve both the apparent FRET efficiency () and for the FRET sample in the presence of free donors and acceptors.11 Both E-FRET and Lux-FRET methods require at least three reference samples separately expressing donor-only, acceptor-only, and donor-acceptor tandem construct for calibrations, and all experiments for references and FRET samples must be performed under the same measurement conditions.9,15,17 E-FRET can be implemented on widefield and confocal microscopes.8,20 Considering the stability of widefield microscopes and the low cost, E-FRET is generally implemented on widefield microscopes.1,9,15,21 However, it is generally difficult to choose an appropriate optical filter as a donor emission channel to selectively collect donor emission, a requirement of an E-FRET method,9,15 for some FPs pairs such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP).22 FRET quantification in single living cells by spFRET is generally implemented on a confocal microscope with spectral detectors (Zeiss 510 META, Zeiss 710/780, Olympus FV 1000/1200, Leica TCS SP2/5/8, and Nikon A1).11 There are two spectral acquisition modes for confocal microscopes:23 (1) synchronization acquisition of multiband spectra with about 10-nm spectral resolution,16,24 which is useful for multispectral imaging of rapid processes in living cells.17 However, fewer data points may limit the accuracy when spectrally unmixing signals from the FRET sample;25 (2) time-lapse acquisition of multiband spectra with about 2-nm spectral resolution,26 which needs a long detection time. In this report, for the first time, we combine a miniature fiber optic spectrometer and a widefield fluorescence microscope to perform spectral measurement of FRET quantification in single living cells. This platform has two specialties: (1) independent excitation and emission controllers; (2) simultaneous microscopic imaging and spectral detection. In addition, we developed a modified spFRET method, mlux-FRET, for quantifying the multiple donors and multiple acceptors system ( construct, represents the number of donors in the FRET construct) in the presence of free donors and acceptors and a spectra-based 2-channels E-FRET (spE-FRET) method for quantifying the FRET construct. Finally, we implemented these modified FRET quantification methods on our platform to measure the absolute FRET efficiency () and acceptor-to-donor concentration ratio () of tandem constructs with different acceptor/donor stoichiometries in single living Huh-7 cells. 2.Experimental SetupFigure 1(a) depicts the schematic of this platform which consists of a widefield fluorescence microscope (Axio Observer, Carl Zeiss, Oberkochen, Germany) and two detection channels: a spectral detection channel and a CCD imaging channel. The excitation light source is a metal halide lamp (X-Cite 120, Carl Zeiss, Oberkochen, Germany), and its intensity can be attenuated in five discrete steps (TC1, transmission controller) (, 12.5%, 25%, 50%, and 100%), and another neutral density filters’ controller (TC2) with six discrete steps (, 20%, 40%, 60%, 80%, and 100%) can be used for the same purpose. Collimated light from the lamp first transmits through an excitation controller (EC) with three filters. In our study, two excitation bandpass filters of BP 405/20 (Ex405 nm) (BP Exciter, Chroma) and BP 436/20 (Ex436 nm) (BP 436/20, Carl Zeiss, Oberkochen, Germany) were installed in the No. 1 and No. 2 positions, respectively, and the No. 3 position is empty. An objective ( oil) is used to focus the illumination light into the sample, and emission is collected through the same objective and sent back into the same dichroic mirror (DM) and a long-pass (LP) filter. We chose here a primary DM of FT 455 (455DCLP Dichroic, Chroma) and an emission LP filter of LP 455 (455DCLP Dichroic, Chroma) as emission control. Fluorescence reflected by the mirror first passes through a detection controller (DC), and then through a cooled CCD camera (AxioCam MRm, Carl Zeiss, Oberkochen, Germany) and/or a miniature fiber optic spectrometer (QE65 Pro, Ocean Optics, Florida). The DC has three detection modes: (1) microscopic imaging by CCD channel that operates as a guidance for finding the cells; (2) spectral detection by spectrometer channel; (3) simultaneous microscopic imaging and spectral detection. For our spectrometer-microscope platform, the EC is independent of the emission controller, thus we can get two different combinations of excitation and emission filters by adjusting EC, which is very important for the rapid measurement of two excitation wavelengths-based spFRET methods. The miniature fiber optic spectrometer (QE65 Pro) is a compact and flexible system without moving parts, and can be readily connected to a widefield microscope with an interfacing connection type C. The detector used in QE65 spectrometer is a back-thinned, element CCD array, and the spectral detection range is 221.705 to 1015.805 nm. Each count() at the emission wavelength recorded by singe column pixels (64 pixels) of the QE65 spectrometer is related to the photons from about a 0.761-nm wavelength range. In our study, the diameter () of the fiber (QP600-2-UV-VIS, Ocean Optics, Florida) is . The effective imaging area of a CCD camera with a pixel resolution of () is about and the corresponding imaging sample area is about . We first installed the same two CCD cameras on the two detection channels to collimate them using a standard fluorescence sample, and then replaced one CCD camera with the interfacing connection type C of the spectrometer. The detection area () of the spectrometer is: corresponding to the of the sample area. Figure 1(b) showed the count spectra of a Huh-7 cell expressing YFP-Bax at different positions (inset figures) with 500-nm excitation. When the cell was centered in the CCD channel, the corresponding fluorescence intensity collected by the spectrometer was the maximum, indicating that the detection area of the spectra channel concurs with the middle of the CCD imaging channel. In our experiments, fluorophores were excited by 405 or 436 nm () excitation. We also used a 500 nm () excitation with the combination of an excitation bandpass filter of BP (Ex500 nm) (BP , Carl Zeiss, Oberkochen, Germany), primary DM FT 515 (BP , Carl Zeiss, Oberkochen, Germany), and emission bandpass filter of BP (BP , Carl Zeiss, Oberkochen, Germany) to bleach the acceptor. The fluorescence intensity is collected in the spectra range of 460 to 620 nm. 3.DNA Constructs and Cells3.1.Reagent and PlasmidsCerulean, Venus-kras, and YFP-Bax plasmids were purchased from Addgene Company (Cambridge, Massachusetts). The FRET-standard constructs, including Cerulean-32-Venus (C32V, Addgene plasmid 29396), Cerulean-5-Venus-5-Cerulean (CVC, Addgene plasmid 27788), Venus-5-Cerulean-5-Venus (VCV, Addgene plasmid 27788), and Venus-5-Cerulean-5-Venus-6-Venus (VCVV, Addgene plasmid 27789), were kindly provided by the Vogel lab (National Institutes of Health, Bethesda, Maryland). 3.2.Cell Culture and TransfectionHuh-7 cells, a human hepatocellular carcinoma cell line, were obtained from the Department of Medicine, Jinan University, Guangzhou, China. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, New York) containing 10% fetal calf serum (FCS; Sijiqing, Hangzhou, China) at 37°C under 5% in a humidified incubator. For transfection, cells were cultured in DMEM containing 10% FCS in a 35-mm glass dish with a density of at 37°C under 5% in a humidified incubator. After 24 h, when the cells reached about 70% to 90% confluence, plasmid was transfected into the Huh-7 cells for 24-48 h. Turbofect™ (Fermentas Inc., Glen Burnie, Maryland) was used as a transfection reagent. 4.FRET Quantification MethodsThroughout the paper, count denotes the measured count spectrum recorded by the spectrometer at specific measurement conditions, and the superscript number denotes the excitation wavelength ; subscript capital letter denotes the experimental sample (“” denotes the donor sample; “” denotes the acceptor sample, and space denotes the FRET sample). 4.1.mlux-FRET: Modified lux-FRET TheoryWith two excitation wavelengths, lux-FRET can simultaneously measure the absolute and of the FRET samples including FRET constructs and free donors and acceptors.11,17 In addition, for the time-invariant and uniform acceptor/donor ratio such as the tandem construct sensors, after an initial spectrally resolved dual-excitation calibration, the user can perform repetitive single-excitation wavelength measurements to quantify at high temporal resolution.11,17 Here, we developed a modified lux-FRET method, mlux-FRET, to measure the absolute and of the FRET samples including FRET constructs, free donors, and acceptors. Considering an FRET sample containing FRET constructs, free donors, and free acceptors, the emission spectra should be a linear combination of five contributions from the free donor, the donor in FRET constructs, the free acceptor and the acceptor in FRET constructs including direct excitation and sensitized emission: where is the transfer factor of the spectrometer from fluorescence intensity to count at the emission wavelength ; is the FRET efficiency of FRET construct; is the illumination intensity at excitation; is the detection efficiency of the instrument used; and are the extinction coefficients of the donor and acceptor at excitation; and are the quantum yields of the donor and acceptor; and are the spectral fingerprints of the donor and acceptor normalized to unit area; [] and [] are the concentrations of free donors and acceptors in the FRET sample; [] is the concentration of constructs.Sorting these terms in Eq. (1) according to those with emission characteristics of the donor and acceptor, respectively, we obtain where and are the reference count spectra obtained from cells separately expressing donors with concentration [] and acceptors with concentration [], respectively, under the same measurement conditions as the FRET samples:We define two apparent relative donor and acceptor concentrations, respectively, as follows: and Eq. (2) becomes:We can obtain the corresponding excitation ratio () at excitation from the reference measurements [Eqs. (3) and (4)] Considering two different excitation wavelengths, Eqs. (5) and (6) represent three independent equations [since Eq. (5) does not rely on the extinction coefficient ratio ]. From Eqs. (5), (6), and (8), we can obtain the apparent efficiency () and : where [] and [] are the concentrations of total acceptors and donors; and are the fractions of the donors and acceptors participating in the FRET complexes, respectively, and the calibration factor can be obtained using a tandem construct with a known acceptor/donor stoichiometry, and .17In fact, the lux-FRET method proposed by Wlodarczyk et al. has the same and equations as Eqs. (9) and (11) except that the equation is different from Eq. (10) and is given as11,17 where [] is the concentration of the pair.4.2.spE-FRET: Spectra-Based 2-Channel E-FRET MethodBased on the measured (, 2) spectra at two different excitations, we developed a spectra-based E-FRET method, spE-FRET, to calculate the and values. was calculated as follows: where is a calibration factor under the emission wavelengths of and ; is the measured count value at emission wavelength with excitation; the superscript SE denotes the sensitized acceptor fluorescence count, and where , , , and are the crosstalk correction coefficients obtained by using donor-only and acceptor-only samples as follows: where the subscript capital letter denotes the sample (“” denotes donor-only sample and “” denotes acceptor-only sample). Once these coefficients are determined, all subsequent experiments must be performed under the same measurement conditions. The calibration factor under this measurement condition can be obtained by using a donor-acceptor tandem construct as follows: where the superscript “post” denotes the corresponding emission count values after selectively photobleaching partial acceptors.We can also measure as follows: where is the calibration factor for , and can be obtained using a tandem construct with a acceptor-donor stoichiometry under the same measurement conditions.155.Results and Discussion5.1.Spectral Fingerprints of Venus and Cerulean in Living Huh-7 CellsIn order to use our platform to measure the spectral fingerprints of Cerulean [] and Venus [] in living Huh-7 cells, we first used a standard lamp to calibrate the instrument. Calibration was performed with a halogen tungsten lamp calibrated by a spectrometer (QE65 Pro, Ocean Optics, Florida) that was calibrated by a standard light source (LS-1-CAL, Ocean Optics, Florida). Figure 2(a) showed the reference spectrum [] of the halogen lamp directly measured by the calibrated QE65 Pro spectrometer, the spectrum [] of the halogen lamp obtained by our spectroscopic widefield fluorescence microscope, and the corresponding spectral sensitivity calibration curve: . The same curves were obtained at an interval of 3 months [Fig. 2(b)], demonstrating the stability of the spectrometer-microscope platform. Figure 2(c) showed the spectra of a representative living Huh-7 cell expressing Cerulean (upper two lines) and the corresponding background from cells without expressing FPs (lower two lines), showing that the autofluorescence and background were very low. The mean spectra from cells without expressing FPs under the same measurement conditions were subtracted from the measured fluorescence spectrum. Although 436-nm excitation excited Cerulean more effectively [Fig. 2(c)], the normalized spectrum [Fig. 2(d), black line] was the same as the normalized spectrum with a 405-nm excitation [Fig. 2(d), dashed line], and this spectrum was used as the spectral fingerprint [] of Cerulean. Similarly, the normalized spectrum of Venus in living Huh-7 cells with 436-nm excitation was used as the spectral fingerprint [] of Venus [Fig. 2(e)]. In reality, and were obtained from at least 15 living cells. Although the measured of Cerulean decreased with the decreasing exposure time, normalization showed the same spectra (data not shown), indicating that the exposure time of spectrometer did not influence the spectral fingerprints. The shortest exposure time of spectrometer is 8 ms, and in this report, the exposure time is 50 ms. The stability of the platform is very important for FRET quantification in living cells. Some FRET quantification methods need to calibrate the instrument setup.9,10,15 For example, three external references are required to determine the instrument setup calibration factor () for the E-FRET method9,15 and the sp-RET method needs to rigorously calibrate the spectral response of the complete optical setup.10 Although some FRET quantification methods do not need to calibrate the optical setup, the correction coefficients for removing spectral crosstalks also depend on the stability of the instrument.11,16,17 All SE-FRET methods need complicated crosstalk correction,9–11,15–19 and these correction coefficients are intrinsic to the given fluorophores and optical setup as well as the illumination.1,9,10,17,21,27 In reality, the fluorophores and illumination light are very stable.9,21,27 Therefore, once these correction coefficients and calibration factors are predetermined for a given choice of fluorophores and instrument, it is not necessary to measure them for every experiment with a steady-state FRET instrument.1,9,10,19,21,27 Although our spectrometer-microscope platform is very stable over at least 3 months [Fig. 2(b)], periodic calibration consistently improves the fit quality and the reliability of the measurements. In reality, calibration of the optical setup is very easy, and the normative calibration can be completed in a few minutes. Since the EC is independent of the emission controller, we, here, detected the two spectra at 405- or 436-nm excitation with the same emission channels. Therefore, we used the same calibration curve to correct the measured spectra with different excitations, which is contrary to the sp-RET method proposed by Levy et al.10 In fact, the calibration step [Fig. 2(a)] is not mandatory. Spectral fingerprints of the donor [] and acceptor [] are independent of instrument and illumination, thus they can also be obtained by using other instruments such as a spectrofluorometer.17 Moreover, once and were determined, we did not need to measure them for every experiment in the specific cell line. In order to insure the accurate measurement of total donor and acceptor emissions, the acquisition range should overlap well with their emission spectra. The spectral ranges chosen in this study fulfill this requirement, covering and of the Cerulean and Venus emission spectra, respectively [Figs. 2(d) and 2(f)]. 5.2.Implementation of mlux-FRET for Measuring the of C32V in Living CellsTo use mlux-FRET to measure the of C32V in living cells, we first measured four reference emission spectra of the Cerulean-only sample and the Venus-only sample, respectively, with two different excitations, and then measured two emission spectra of the C32V sample at the two different excitations under the same measurement conditions. Figure 3(a) showed the images of cells separately expressing Cerulean and Venus with 405-nm () and 436-nm () excitations, respectively, and the reference spectra inside the living cell indicated by the red circles , and , were shown in Figure 3(b). Excitation ratios were determined from the above spectra as: [Fig. 3(c)] and [Fig. 3(d)], using quantum yield values of donor and acceptor, and .28 Figure 3(e) showed the images of representative cells expressing C32V under the same measurement conditions as Fig. 3(a), and the spectra and mlux-FRET unmixing (black line) for the live cell indicated by red circles were shown in Figs. 3(f) and 3(g). The estimated parameters were , , and . Substitute , , , and values into Eqs. (9) to (11) to obtain and . The statistical results from at least 13 cells expressing C32V were and . In reality, we must be very careful when obtaining the reference spectra of Venus with 405-nm excitation due to the very low extinction coefficients of Venus at 405 nm [Fig. 3(a)]. Although we can use strong 405-nm excitation to excite Venus, this strong illumination significantly bleached the Venus in C32V (data not shown). Therefore, we must choose cells expressing a high level of Venus as a reference. Although Wlodarczyk et al. used 458- and 488-nm excitations to perform lux-FRET for ECFP-EYFP pair,17 it is very hard to use a 488 nm laser line to excite Cerulean or ECFP,10 which was confirmed by the fact that the (488-nm excitation) was 0.02, which is much less than the (458-nm excitation).17 For 458- and 488-nm excitations, an alternative choice is to use GFP as a donor and YFP or Venus as an acceptor for the implementation of mlux-FRET on a confocal microscope. The acceptor cross-excitation fraction is intrinsic to the extinction coefficient ratio () of the donor to acceptor and their relative concentrations. Different at two different excitations is necessary for the mlux-FRET method according to Eq. (4). Although the two ratios (, 2) are related to the cell lines to some extent, they depend mainly on the excitation wavelengths intrinsic to the excitation light source and the transmission properties of the instrument for a given donor-acceptor pair. 405-nm excitation can effectively excite Cerulean/CFP but can hardly excite Venus/YFP, 458-nm excitation can effectively excite both Cerulean/CFP and Venus/YFP, and 488-nm excitation can effectively excite Venus/YFP but hardly excite Cerulean/CFP. Although the ratio with 458- and 488-nm excitations is 114.5,9 it is inapplicable to the CFP (Cerulean)–YFP (Venus) pair due to the very low extinction coefficient of CFP (Cerulean) at the 488-nm wavelength. For our spectrometer-microscope platform, 405- and 436-nm excitations () induced significantly different spectra for C32V, CVC, VCV, and VCVV constructs [Figs. 5(b)–5(e)]. Another better choice, in this view, is the 430- and 445-nm excitations for the CFP (Cerulean)–YFP (Venus) pair. 5.3.Implementation of spE-FRET for Measuring the E of C32V in Living CellsFor spE-FRET, we first calculated the spectral crosstalk coefficients according to Eq. (15). We choose 470.107 to 470.883 nm emission data as and 530.452 to 531.223 nm emission data as . From the measured spectra in Fig. 3(b), the crosstalk correction coefficients for Cerulean and Venus on our spectrometer-microscope platform were , , , and . In order to determine the parameter , we next used a strong 500-nm excitation to selectively bleach partial acceptors inside the same cell as that in Fig. 3(e). Figure 4(a) showed the images of cells in Fig. 3(e) before and after partially photobleaching the acceptor, and the corresponding count spectra of the cell indicated by the red circle in Fig. 4(a) were shown in Fig. 4(b). The degree of PbFRET () was 35.46% [inset figure in Fig. 4(b)]. Substitute , , , , , and [Fig. 4(b)] into Eq. (16) to obtain , and the statistical from at least 13 living cells. Substitute and these count values into Eqs. (13) and (17) to obtain and for the cells indicated by the red circle in Fig. 4(a), and the statistical results from 13 living cells were and . With spE-FRET, the sensitized fluorescence is calculated by subtraction of the major crosstalk components from , and the minor crosstalk components from and according to Eq. (14), which is applicable only when the donor channel selectively or mainly collects donor emission.9 For to 510 nm and to 561.221 nm, , , , and , where is not ignorable, and the corresponding from at least 12 living cells and , which is much less than 27.75%, further demonstrating our recent study that spE-FRET is also very sensitive to the acceptor emission bleedthrough to the donor channel.24 It is generally difficult to choose an appropriate optical filter as a donor emission channel to selectively collect donor emission on a conventional widefield fluorescence microscope.9,22 However, it is very easy to choose a wideband (525 to 535 nm) and a narrowband (476 to 476.69 nm) to make the crosstalk coefficient negligible for the Cerulean-Venus pair on our spectrometer-microscope platform. Moreover, choosing 485.624 to 486.399 nm emission data as , and 520.426 to 550.466 nm emission data as for the GFP–Venus pair, the corresponding is also negligible. 5.4.Quantifying Tandem Constructs with Different Acceptor/Donor Stoichiometries in Single Living Huh-7 Cells Using mlux-FRET and spE-FRET MethodsWe next used mlux-FRET and spE-FRET methods to measure the values of C32V, CVC, VCV, and VCVV constructs inside single living Huh-7 cells. For a live cell expressing the FRET tandem construct, we first measured the spectra at two different excitations. Figure 5(a) showed the images of representative cells expressing C32V, CVC, VCV, and VCVV, respectively, under the same measurement conditions as the Cerulean-only and Venus-only samples in Fig. 3(a). The corresponding normalized spectra of the cell indicated by the red circle in Fig. 5(a) with excitation and the corresponding values calculated by mlux-FRET and spE-FRET, respectively, were shown in Figs. 5(b)–5(e). The statistical values from at least 16 live Huh-7 cells in four independent experiments calculated by the two methods showed consistent results for C32V, CVC, VCV, and VCVV constructs, respectively [Fig. 5(f)], and the corresponding values calculated by spE-FRET and mlux-FRET methods were shown in Fig. 5(g). The formalism [Eq. (10)] of mlux-FRET is different from that [Eq. (12)] of lux-FRET. As shown in Fig. 5(h), the values calculated by Eq. (10) were consistent with the values, while the values calculated by Eq. (12) were inconsistent with the values except for the C32V construct, verifying the ability of mlux-FRET methods to determine the for system. In fact, both the mlux-FRET and spE-FRET methods can be used to measure and values of the construct where the “” and “” are unknown. However, a mlux-FRET method can be used to measure the value of construct only when the ratio is known. In reality, the relative concentration of each molecule contained in a biological complex can be predetermined by biotechnology such as flow cytometry, mass spectrometry, or western blotting.14 6.ConclusionIn this paper, we developed a spectrometer-microscope platform for simultaneous microscopic imaging and spectral detection in single living cells. The independent excitation and emission controllers make this platform very applicable to implement the most commonly used FRET quantification methods including spFRET and SE-FRET quantification methods in single living cells. A complete FRET quantification measurement on our spectrometer-microscope platform can be completed within 1 to 2 s, and the speed of the current configuration is mainly limited by the manual switch of the two excitation filters. With a programmable excitation and DC system, this platform can monitor the dynamic event inside single living cells on a millisecond scale. In reality, the EC with three filters of this spectrometer-microscope platform can be replaced with a filter wheel with six filters, then the spFRET method based upon the simultaneous unmixing of both excitation and emission spectra18,19,29 can be readily implemented on this platform. We also developed a modified spFRET method, mlux-FRET, for quantifying the construct sample in the presence of free donors and acceptors. Moreover, the spE-FRET method we developed here can be readily implemented on this spectrometer-microscope platform for paired FPs with a large spectral overlap such as the GFP-YFP pair. AcknowledgmentsThe authors thank Professor S.S. Vogel (NIH/NIAAA) for providing C32V, CVC, VCV, and VCVV plasmids. This work is supported by the National Natural Science Foundation of China (NSFC) (Grant Nos. 81471699 and 61178078) and Guangzhou Science and Technology Plan Project (2014J4100055). ReferencesT. Zal and N. R. J. Gascoigne,
“Photobleaching-corrected FRET efficiency imaging of live cells,”
Biophys. J., 86
(6), 3923
–3939
(2004). http://dx.doi.org/10.1529/biophysj.103.022087 BIOJAU 0006-3495 Google Scholar
I. L. Medintz et al.,
“Recent progress in developing FRET-based intracellular sensors for the detection of small molecule nutrients and ligands,”
Trends Biotechnol., 24
(12), 539
–542
(2006). http://dx.doi.org/10.1016/j.tibtech.2006.10.008 TRBIDM 0167-7799 Google Scholar
B. P. Maliwal et al.,
“Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors,”
J. Biomed. Opt., 17
(1), 011006
(2012). http://dx.doi.org/10.1117/1.JBO.17.1.011006 JBOPFO 1083-3668 Google Scholar
H. N. Yu et al.,
“Ma-PbFRET: multiple acceptors FRET measurement based on partial acceptor photobleaching,”
Microsc. Microanal., 19
(1), 171
–179
(2013). http://dx.doi.org/10.1017/S1431927612014079 MIMIF7 1431-9276 Google Scholar
A. l. Fábián et al.,
“Strength in numbers: effects of acceptor abundance on FRET efficiency,”
ChemPhysChem, 11
(17), 3713
–3721
(2010). http://dx.doi.org/10.1002/cphc.201000568 CPCHFT 1439-4235 Google Scholar
P. Bojarski et al.,
“Long-distance FRET analysis: a Monte Carlo simulation study,”
J. Phys. Chem. B, 115
(33), 10120
–10125
(2011). http://dx.doi.org/10.1021/jp202152m JPCBFK 1520-6106 Google Scholar
S. V. Koushik, P. S. Blank and S. S. Vogel,
“Anomalous surplus energy transfer observed with multiple FRET acceptors,”
PLoS One, 4
(11), e8031
(2009). http://dx.doi.org/10.1371/journal.pone.0008031 1932-6203 Google Scholar
A. D. Elder et al.,
“A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission,”
J. R. Soc. Interface, 6
(S1), S59
–S81
(2009). http://dx.doi.org/10.1098/rsif.2008.0381.focus 1742-5689 Google Scholar
T. Zal and N. R. J. Gascoigne,
“Photobleaching-corrected FRET efficiency imaging of live cells,”
Biophys. J., 86
(6), 3923
–3939
(2004). http://dx.doi.org/10.1529/biophysj.103.022087 BIOJAU 0006-3495 Google Scholar
S. Levy et al.,
“SpRET: high sensitive and reliable spectral measurement of absolute FRET efficiency,”
Microsc. Microanal., 17 176
–190
(2011). http://dx.doi.org/10.1017/S1431927610094493 MIMIF7 1431-9276 Google Scholar
A. Zeug et al.,
“Quantitative intensity-based FRET approaches-a comparative snapshot,”
Biophys. J., 103
(9), 1821
–1827
(2012). http://dx.doi.org/10.1016/j.bpj.2012.09.031 BIOJAU 0006-3495 Google Scholar
H. E. Grecco, P. R. Navarro and P. J. Verveer,
“Global analysis of time correlated single photon counting FRET-FLIM data,”
Opt. Express, 17
(8), 6493
–6508
(2009). http://dx.doi.org/10.1364/OE.17.006493 OPEXFF 1094-4087 Google Scholar
H. N. Yu et al.,
“An empirical quantitative FRET method for multiple acceptors based on partial acceptor photobleaching,”
Appl. Phys. Lett., 100
(25), 253701
(2012). http://dx.doi.org/10.1063/1.4729481 APPLAB 0003-6951 Google Scholar
L. L. Zhang et al.,
“Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor,”
Appl. Phys. Lett., 104 243706
(2014). http://dx.doi.org/10.1063/1.4885084 APPLAB 0003-6951 Google Scholar
H. M. Chen et al.,
“Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,”
Biophys. J., 91
(5), L39
–L41
(2006). http://dx.doi.org/10.1529/biophysj.106.088773 BIOJAU 0006-3495 Google Scholar
C. Thaler et al.,
“Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,”
Biophys. J., 89
(4), 2736
–2749
(2005). http://dx.doi.org/10.1529/biophysj.105.061853 BIOJAU 0006-3495 Google Scholar
J. Wlodarczyk et al.,
“Analysis of FRET signals in the presence of free donor and acceptor,”
Biophys. J., 94
(3), 986
–1000
(2008). http://dx.doi.org/10.1529/biophysj.107.111773 BIOJAU 0006-3495 Google Scholar
S. Mustafa et al.,
“Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,”
J. Biomed. Opt., 18
(2), 026024
(2013). http://dx.doi.org/10.1117/1.JBO.18.2.026024 JBOPFO 1083-3668 Google Scholar
A. D. Hoppe et al.,
“N-way FRET microscopy of multiple protein-protein interactions in live cells,”
PLoS One, 8
(6), e64760
(2013). http://dx.doi.org/10.1371/journal.pone.0064760 1932-6203 Google Scholar
H. Düssmann et al.,
“Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,”
Cell Death Differ., 17
(2), 278
–290
(2010). http://dx.doi.org/10.1038/cdd.2009.123 CDDIEK 1350-9047 Google Scholar
A. D. Hoppe, K. Christensen and J. A. Swanson,
“Fluorescence resonance energy transfer-based stoichiometry in living cells,”
Biophys. J., 83
(6), 3652
–3664
(2002). http://dx.doi.org/10.1016/S0006-3495(02)75365-4 BIOJAU 0006-3495 Google Scholar
T. Zimmermann et al.,
“Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,”
FEBS Lett., 531 245
–249
(2002). http://dx.doi.org/10.1016/S0014-5793(02)03508-1 FEBLAL 0014-5793 Google Scholar
Y. Hiraoka, T. Shimi and T. Haraguchi,
“Multispectral imaging fluorescence microscopy for living cells,”
Cell Struct. Funct., 27
(5), 367
–374
(2002). http://dx.doi.org/10.1247/csf.27.367 CSFUDY 0386-7196 Google Scholar
H. L. Li, H. N. Yu and T. S. Chen,
“Partial acceptor photobleaching-based quantitative FRET method completely overcoming emission spectral crosstalks,”
Microsc. Microanal., 18
(5), 1021
–1029
(2012). http://dx.doi.org/10.1017/S1431927612001110 MIMIF7 1431-9276 Google Scholar
L. Peng et al.,
“Fourier fluorescence spectrometer for excitation emission matrix measurement,”
Opt. Express, 16
(14), 10493
–10500
(2008). http://dx.doi.org/10.1364/OE.16.010493 OPEXFF 1094-4087 Google Scholar
T. J. Chancellor et al.,
“Actomyosin tension exerted on the nucleus through nesprin-1 connections influences endothelial cell adhesion, migration, and cyclic strain-induced reorientation,”
Biophys. J., 99
(1), 115
–123
(2010). http://dx.doi.org/10.1016/j.bpj.2010.04.011 BIOJAU 0006-3495 Google Scholar
T. Zal, M. A. Zal and N. R. Gascoigne,
“Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse,”
Immunity, 16
(4), 521
–534
(2002). http://dx.doi.org/10.1016/S1074-7613(02)00301-1 IUNIEH 1074-7613 Google Scholar
N. C. Shaner, P. A. Steinbach and R. Y. Tsien,
“A guide to choosing fluorescent proteins,”
Nat. Methods, 2
(12), 905
–909
(2005). http://dx.doi.org/10.1038/nmeth819 1548-7091 Google Scholar
J. Yuan et al.,
“Quantitative FRET measurement by high-speed fluorescence excitation and emission spectrometer,”
Opt. Express, 18
(18), 18839
–18851
(2010). http://dx.doi.org/10.1364/OE.18.018839 OPEXFF 1094-4087 Google Scholar
BiographyLiuying Chai received a BS degree in physics from Anyang Normal University, China, in 2012. Her current research interests include fluorescence microscopy and biomedical optics. Jianwei Zhang received an MS degree in optics from South China Normal University, China. He has three years of experience in fluorescence microscopy and biomedical optics. He is the author of two journal papers. Lili Zhang received a BS degree in physics from Hubei University, China. She is the author of one journal paper. Her current research interests include fluorescence microscopy and biomedical optics. Tongsheng Chen received a PhD degree in biomedical engineering from Huazhong University of Science and Technology, China. He has 15 years of experience in fluorescence microscopy and biomedical optics. Since 2005, he has been a full professor of MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou, China. He is the author of more than 100 journal papers, and he is also a member of SPIE. |