Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
- Universität Potsdam, Institute of Biochemistry and Biology, Germany
Figures
Figure 1 with 3 supplements
Cross-correlation and signal-to-noise ratio (SNR) analysis for two-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements at the plasma membrane (PM) of HEK 293T cells, performed with fluorescent proteins (FPs) showing strongly overlapping emission spectra.
(A) Representative correlation functions (CFs) (green: autocorrelation function [ACF] for mEGFP [‘G’]; yellow: ACF for mEYFP [‘Y’] gray: cross-correlation function [CCF] calculated for both fluorophore species) obtained from SFSCS measurements on the PM of HEK 293T cells co-expressing mp-mEGFP and mp-mEYFP. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (B) Relative cross-correlation values obtained from SFSCS measurements described in (A) (‘G + Y’) or on HEK 293T cells expressing mp-mEYFP-mEGFP heterodimers (‘Y-G’). (C) SNR of ACFs for mEGFP (green) and mEYFP (yellow), obtained from SFSCS measurements described in (A), plotted as a function of the average ratio of detected mEGFP and mEYFP fluorescence. (D) Representative CFs (light red: ACF for mApple [‘A’]; dark red: ACF for mCherry2 [‘Ch2’]; gray: CCF calculated for both fluorophores) obtained from SFSCS measurements on the PM of HEK 293T cells co-expressing mp-mApple and mp-mCherry2. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (E) Relative cross-correlation values obtained from SFSCS measurements described in (D) (‘A + Ch2’) or on HEK 293T cells expressing mp-mCherry2-mApple heterodimers (‘Ch2-A’). (F) SNR of ACFs for mApple (light red) and mCherry2 (dark red), obtained from SFSCS measurements described in (D), plotted as a function of the average ratio of detected mApple and mCherry2 fluorescence. Data are pooled from three (B) or two (E) independent experiments each. The number of cells measured is given in parentheses. Error bars represent mean ± SD.
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Figure 1—source data 1
Relative cross-correlation and signal-to-noise ratios for two-species scanning fluorescence correlation spectroscopy measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Fluorescent protein (FP) emission spectra.
Average emission spectra of mp-mEGFP, mp-mEYFP, mp-mApple, and mp-mCherry2 measured by spectral imaging (23 spectral channels from 491 nm to 695 nm) with 488 nm and 561 nm excitation on HEK 293T cells expressing each FP individually. Spectra are shown for two different days (day 1: solid line; day 2: dotted line) and averaged over four cells each. For each cell, 25 frames were acquired and pixels corresponding to the plasma membrane (PM) semi-manually segmented in the average image (manual selection followed by removal of pixels with intensities below 25% of the maximum pixel intensity in the selected region).
Figure 1—figure supplement 2
Spectral filters for two-species scanning fluorescence spectral correlation spectroscopy (SFSCS).
(A, B) Photon weights calculated in spectral decomposition of SFSCS data acquired on HEK 293T cells expressing mp-mEYFP-mEGFP (A) or mp-mCherry2-mApple (B).
Figure 1—figure supplement 3
Scanning fluorescence spectral correlation spectroscopy (SFSCS) on fluorescent protein (FP) heterodimers.
(A) Representative correlation functions (CFs) (green: autocorrelation function [ACF] for mEGFP [‘G’]; yellow: ACF for mEYFP [‘Y’]; gray: cross-correlation function [CCF] calculated between both fluorophore signals) obtained from SFSCS measurements on the plasma membrane (PM) of living HEK 293T cells expressing mp-mEYFP-mEGFP heterodimers. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (B) Representative CFs (light red: ACF for mApple [‘A’]; dark red: ACF for mCherry2 [‘Ch2’]; gray: CCF calculated between both fluorophore signals) obtained from SFSCS measurements on the PM of living HEK 293T cells expressing mp-mCherry2-mApple heterodimers. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (C) Relative cross-correlation values obtained from SFSCS measurements on HEK 293T cells expressing mp-mEYFP-mEGFP (rigid linker between the two FPs, see Supplementary file 1a) or mp-mEGFP-mEYFP (short linker between the two FPs, Supplementary file 1a) heterodimers. Data are pooled from three independent experiments each. The number of cells measured is given in parentheses. Error bars represent mean ± SD. Statistical significance was determined using Welch’s corrected two-tailed Student’s t-test (**p<0.05).
Figure 2 with 1 supplement
Diffusion and molecular brightness analysis for two-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements at the plasma membrane (PM) of HEK 293T cells.
(A) Diffusion times obtained from SFSCS measurements on HEK 293T cells expressing either influenza A virus (IAV) HA-mEGFP or mp-mEYFP separately (blue), or co-expressing both fusion proteins (red). (B) Normalized molecular brightness values obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP and mp-mEYFP (blue), mp-2x-mEGFP and mp-mEYFP (red), or expressing mp-mEGFP alone (yellow). Normalized brightness values were calculated by dividing molecular brightness values detected in each SFSCS measurement by the average brightness obtained for mEGFP and mEYFP in cells co-expressing mp-mEGFP and mp-mEYFP. Data are pooled from two independent experiments for each sample. The number of cells measured is given in parentheses. Error bars represent mean ± SD. Statistical significance was determined using Welch’s corrected two-tailed Student’s t-test (****p<0.0001, ns: not significant).
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Figure 2—source data 1
Diffusion times and normalized molecular brightness values for two-species scanning fluorescence correlation spectroscopy measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Relative cross-correlation obtained from two-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements.
The number of cells measured is given in parentheses. Error bars represent mean ± SD.
Figure 3 with 4 supplements
Cross-correlation and molecular brightness analysis for three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements on fluorescent protein (FP) hetero-oligomers and influenza A virus (IAV) M2 at the plasma membrane (PM) of HEK 293T cells.
(A–C) Representative correlation functions (CFs) (green/yellow/red: autocorrelation functions [ACFs] for mEGFP [‘G’]/mEYFP [‘Y’]/mCherry2 [‘Ch2’]; purple/blue/gray: cross-correlation functions [CCFs] calculated for the pairs mEGFP and mEYFP/mEGFP and mCherry2/mEYFP and mCherry2) obtained from three-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mCherry2 (A), mp-mCherry2-mEGFP heterodimers and mp-mEYFP (B), or expressing mp-mEYFP-mCherry2-mEGFP heterotrimers (C), as illustrated in insets. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (D) Representative fluorescence images of HEK 293T cells co-expressing CD9-mEGFP, LC3-mEYFP, and IAV protein M2-mCh2. Spectral filtering and decomposition were performed to obtain a single image for each species. Scale bars are 5 µm. (E) Representative CFs (green/yellow/red: ACFs for mEGFP/mEYFP/mCherry2; purple/blue/gray: CCFs calculated for the pairs mEGFP and mEYFP/mEGFP and mCherry2/mEYFP and mCherry2) obtained from three-species SFSCS measurements on HEK 293T cells co-expressing CD9-mEGFP, LC3-mEYFP, and M2-mCh2. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (F) Relative cross-correlation values obtained from three-species SFSCS measurements described in (A–C) and (E). (G) Normalized molecular brightness values obtained from three-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2 (blue), mp-2x-mEGFP, mp-mEYFP, and mp-mCherry2 (red), CD9-mEGFP, LC3-mEYFP, and M2-mCh2 (green), or expressing mp-mEGFP alone (yellow). Normalized brightness values were calculated by dividing the molecular brightness values detected in each SFSCS measurement by the average brightness obtained for mEGFP, mEYFP, and mCherry2 in cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2. Data are pooled from two independent experiments for each sample. The number of cells measured is given in parentheses. Error bars represent mean ± SD.
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Figure 3—source data 1
Relative cross-correlation and normalized molecular brightness values for three-species scanning fluorescence correlation spectroscopy measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Spectral filters for three-species scanning fluorescence spectral correlation spectroscopy (SFSCS).
Photon weights calculated in spectral decomposition of SFSCS data acquired on HEK 293T cells expressing mp-mEYFP-mCherry2-mEGFP. Shown are average photon weights from five SFSCS acquisitions each.
Figure 3—figure supplement 2
Relative cross-correlation (rel.cc.) for three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) analyzed using different fitting algorithms.
Cross-correlation functions (CCFs) obtained from measurements on cells co-expressing mp-mCherry2-mEGFP heterodimers and mp-mEYFP were fitted using as start parameter for the amplitude either a positive value (same for all CCFs, fit routine 1), or the average of the first five points of each CCF (fit routine 2). For non-correlated data (e.g., G,Y and Y,Ch2 combinations), the second fit routine may converge to negative fit amplitudes, resulting in a distribution of rel.cc. values scattered around 0. Fit routine 1 always converged to positive amplitude values, producing low but positive rel.cc. values. Filtering based on the cross-correlation diffusion time (fit routine 1, filtered) removes some of the residual positive rel.cc. in non-correlated data. Here, a threshold value of five times the maximum of the two diffusion times obtained from autocorrelation functions (ACFs) for each respective fluorescent protein (FP) combination was chosen. For correlated data, for example, G,Ch2, both fit routines converged to comparable positive values.
Figure 3—figure supplement 3
Noise analysis of three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements.
Signal-to-noise ratio (SNR) (color coded) of autocorrelation functions (ACFs) for mEGFP (A), mEYFP (B), and mCherry2 (C) channels obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2 as a function of their signal relative to that of the other two fluorescent protein (FP) species. Data was pooled from two independent experiments in which 31 cells were measured in total.
Figure 3—figure supplement 4
Membrane recruitment of LC3 in M2-expressing cells.
(A, B) Fluorescence images of LC3-mEYFP (A) and M2-mCherry2 (B) excited with either 488 nm (A) or 561 nm (B) excitation. LC3 is recruited to the plasma membrane (PM) in cells showing higher expression of M2 (top cell) relative to M2, but remains in the cytosol in cells expressing only low levels of M2 compared to LC3 (bottom cell). Scale bars are 10 µm. (C) Molecular brightness of LC3-mEYFP obtained from three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements shown in Figure 3 as a function of the ratio of LC3-mEYFP to M2-mCherry2 expression at the PM, in units of protein monomers. The number of monomers was calculated by dividing the signal detected for LC3-mEYFP/M2-mCherry2 in SFSCS measurements by the average molecular brightness detected for mEYFP and mCherry2 fluorophores in the monomeric reference sample (cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2, Figure 3).
Figure 4 with 2 supplements
Cross-correlation analysis for four-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements on fluorescent protein (FP) hetero-oligomers in HEK 293T cells.
(A–C) Representative autocorrelation functions (ACFs) (green/yellow/orange/red for mEGFP [‘G’]/mEYFP [‘Y’]/mApple [‘A’]/mCherry2 [‘Ch2’]) obtained from four-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, mp-mApple, and mp-mCherry2 (A), mp-mCherry2-mEGFP heterodimers, mp-mEYFP, and mp-mApple (B), or expressing mp-mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (C), as illustrated in insets. Solid thick lines show fits of a two-dimensional diffusion model to the correlation functions (CFs). (D–F) SFSCS cross-correlation functions (CCFs) (dark blue/ light blue/orange/yellow/red/dark red for CCFs calculated for mEGFP and mEYFP/mEGFP and mApple/mEGFP and mCherry2/mEYFP and mApple/mEYFP and mCherry2/mApple and mCherry2) from measurements described in (A–C) (CCFs in (D)/(E)/(F)) corresponding to ACFs shown in (A)/(B)/(C). Solid thick lines show fits of a two-dimensional diffusion model to the CFs. (G) Relative cross-correlation values obtained from four-species SFSCS measurements described in (A–C). Data are pooled from two independent experiments. The number of cells measured is given in parentheses. Error bars represent mean ± SD.
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Figure 4—source data 1
Relative cross-correlation values for four-species scanning fluorescence correlation spectroscopy measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Spectral filters for four-species scanning fluorescence spectral correlation spectroscopy (SFSCS).
Photon weights calculated in spectral decomposition of SFSCS data acquired on HEK 293T cells expressing mp-mEYFP-mCherry2-mEGFP-mApple. Shown are average photon weights from five SFSCS acquisitions each.
Figure 4—figure supplement 2
Diffusion dynamics of four-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements.
Diffusion times obtained from four-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, mp-mApple, and mCherry2 (blue), mp-mCherry2-mEGFP heterodimers, mp-mEYFP, and mp-mApple (red), or expressing mp-mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (yellow). The four fluorescent protein (FP) species are denoted with ‘G,’ ‘Y,’ ‘A,’ and ‘Ch2.’ Data are pooled from two independent experiments. The number of cells measured is given in parentheses. Error bars represent mean ± SD.
Figure 5 with 2 supplements
Cross-correlation analysis for four-species raster spectral image correlation spectroscopy (RSICS) measurements on fluorescent protein (FP) hetero-oligomers expressed in cytoplasm of A549 cells.
(A–F) Representative RSICS spatial autocorrelation functions (ACFs) (A, C, E) and cross-correlation functions (CCFs) (B, D, F) obtained from four-species RSICS measurements on A549 cells. Cells were co-expressing mEGFP (‘G’), mEYFP (‘Y’), mApple (‘A’), mCherry2 (‘Ch2’) (A, B), mCherry2-mEGFP and mEYFP-mApple heterodimers (C, D), or mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (E, F). (G, H) Relative cross-correlation values (G) and diffusion coefficients (H) obtained from four-species RSICS measurements described in (A–F). Data are pooled from two independent experiments. The number of cells measured is given in parentheses. Error bars represent mean ± SD.
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Figure 5—source data 1
Relative cross-correlation values and diffusion coefficients for four-species raster spectral image correlation spectroscopy measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Fluorescent protein (FP) emission spectra.
Average emission spectra measured on HEK 293T cell samples (solid line) described in Figure 1—figure supplement 1 or on A549 cells expressing cytosolic mEGFP, mEYFP, mApple, and mCherry2 (dotted line). Spectra measured on four cells each were averaged over 3 (HEK 293T) or 2 (A549) days. For A549 cells, a homogeneous region of interest (ROI) in the cytosol was manually selected.
Figure 5—figure supplement 2
Fluorescent protein (FP) emission spectra at different pH values.
(A–D) Average emission spectra of GPI-mEGFP (A), GPI-mEYFP (B), GPI-mApple (C), and GPI-mCherry2 (D) measured by spectral imaging (23 spectral channels from 491 nm to 695 nm) using 488 nm and 561 nm excitation on HEK 293T cells supplemented with buffer at different pH values, ranging from pH 5.0 to pH 9.2. At each pH value, ca. 10–20 cells were imaged for five frames. To obtain average emission spectra, pixels corresponding to the plasma membrane (PM) were semi-manually segmented (manual selection followed by removal of pixels with intensities below 25% of the maximum pixel intensity in the selected region) and detected spectra averaged over all pixels and cells measured at each pH.
Figure 6 with 1 supplement
Three-species raster spectral image correlation spectroscopy (RSICS) measurements on influenza A virus (IAV) polymerase complex (PC) and fluorescent protein (FP) hetero-oligomers in the nucleus of A549 cells.
(A) Representative fluorescence image (left) of A549 cells co-expressing FP-tagged IAV PC proteins PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. Spectral filtering and decomposition result in a single image for each species (right), denoted with ‘Y,’ ‘G,’ and ‘Ch2.’ Scale bars are 10 µm. (B, C) Representative RSICS spatial autocorrelation functions (ACFs) (B) and cross-correlation functions (CCFs) (C) obtained from three-species RSICS measurements on A549 cells co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. (D) Relative cross-correlation values obtained from three-species RSICS measurements on A549 cells co-expressing mEGFP, mEYFP, and mCherry2 (blue), PA-mEYFP, PB1-mEGFP, PB2-mCherry2 (green), or expressing mEYFP-mCherry2-mEGFP heterotrimers (red). Data are pooled from four independent experiments. (E) Normalized molecular brightness values obtained from three-species RSICS measurements on A549 cells co-expressing mEGFP, mEYFP, and mCherry2 (blue), 2x-mEGFP, mEYFP, and mCherry2 (red), 2x-mEGFP, 2x-mEYFP, 2x-mCherry2 (yellow), or PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 (green). Data are pooled from three (2x-mEGFP + mEYFP + mCherry2, 2x-mEGFP + 2x-mEYFP + 2x-mCherry2), four (PA-mEYFP + PB1-mEGFP + PB2-mCherry2), or five (mEGFP + mEYFP + mCherry2) independent experiments. (F) Diffusion coefficients obtained from three-species RSICS measurements on A549 cells co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. Data are pooled from four independent experiments. For (D–F), the number of cells measured is given in parentheses. Error bars represent mean ± SD.
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Figure 6—source data 1
Relative cross-correlation, normalized molecular brightness values, and diffusion coefficients for three-species raster spectral image correlation spectroscopy measurements on influenza A virus complex and fluorescent protein hetero-oligomers in the nucleus of A549 cells.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Cross-correlation and diffusion analysis for three-species raster spectral image correlation spectroscopy (RSICS) measurements on influenza A virus (IAV) polymerase complex as a function of relative protein concentration.
(A–C) Relative cross-correlation for PA-mEYFP and PB2-mCherry2 (A), normalized molecular brightness (B), and diffusion coefficient (C) detected for PA-mEYFP, obtained from three-species RSICS measurements on A549 cells co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. Data are plotted as a function of the ratio of PB1-mEGFP to PA-mEYFP, in units of protein monomers, and pooled from four independent experiments (n = 53 cells). The number of monomers was calculated by dividing the signal detected for PB1-mEGFP and PA-mEYFP in scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements by the average molecular brightness detected for mEGFP and mEYFP fluorophores in the monomeric reference sample (cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2).
Figure 7
Triple raster image correlation spectroscopy (TRICS) reveals the formation of ternary influenza A virus (IAV) polymerase hetero-complexes in the nucleus of A549 cells.
(A, B) Representative triple-correlation functions (3CFs) obtained from TRICS measurements on A549 cells co-expressing mEGFP, mEYFP, and mCherry2 (‘neg.’) (A) or co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 (‘polym.’) (B). The axes a and b indicate shifts in the x and y direction, respectively, across the three detection channels, as described in Materials and methods. (C) Relative triple-correlation (rel.3C.) values obtained from the measurements described in (A, B). The number of cells measured is given in parentheses. Error bars represent mean ± SD. Statistical significance was determined using Welch’s corrected two-tailed Student’s t-test (****p<0.0001).
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Figure 7—source data 1
Relative triple correlation values for triple raster image correlation spectroscopy analysis of influenza A virus polymerase complex in the nucleus of A549 cells.
- https://cdn.elifesciences.org/articles/69687/elife-69687-fig7-data1-v2.xlsx
Appendix 1—figure 1
Effect of high-pass filter on calculation of variance and third central moment of random numbers sampled from a Poissonian probability distribution.
Variance (f2, blue circles) and third central moment (f3, blue circles) were calculated with a moving average (window size ) for a set of 105 random numbers drawn from a Poissonian distribution with average 10. An empirical function (red solid line) of the form was fitted to the variance (f2) and third central moment (f3), and used to correct for the undersampling effect. The corresponding values after applying the empirical correction are shown as blue circles in the panels labeled as ‘corrected.’
Appendix 1—figure 2
Triple raster image correlation spectroscopy (TRICS) analysis of simulated three-species RICS data.
(A, B) Two-dimensional representation of the triple-correlation function (3CF) calculated for simulated TRICS data (with a four-frame high-pass filter) for (A) ternary hetero-complexes or (B) the same number of particles per species diffusing as independent monomers. From a linear interpolation of G3C to (0,0) (using the first point G3C(1,1) and the average of the four points G3C(1,2), G3C(2,1), G3C(2,2), G3C(3,0)) an approximate value of the 3CF amplitude was determined and corrected with the correction factor discussed in Section A1.1. The obtained value and the autocorrelation function (ACF) amplitude value (also corrected for the decay induced by the high-pass filter) were used to calculate the relative triple-correlation value rel.3C. (given as inset).
Author response image 1
Example of a three-species SFSCS analysis for a measurements on cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2 with variable time binning of [1,2,4,8] x T, acquired with a line-scan time of T~0.4 ms.
Author response image 2
Relative cross-correlation for three-species SFSCS experiments described in Figure 3 of the manuscript, analyzed using different fit routines.
CCFs obtained from measurements on cells co-expressing mp-Cherry2-mEGFP hetero-dimers and mp-mEYFP were either fitted with the same positive initial value for the amplitude (fit routine 1), or with the average of the first five points of each CCF (fit routine 2). For non-correlated data (e.g. G,Y and Y,Ch2 combinations), the second fit routine may converge to negative fit amplitudes, resulting in a distribution of rel.cc. values scattered around 0. Fit routine 1 always converged to positive amplitude values, causing low residual false-positive rel.cc. values. Filtering based on “unreasonable” cross-correlation diffusion time may remove some of the residual positive rel.cc. (fit routine 1, filtered). Here a threshold value of five times the maximum of the two diffusion times obtained from ACFs for each respective FP combination was chosen. For correlated data, e.g. G,Ch2, both fit routines converged to comparable positive values.
Author response image 3
Molecular brightness obtained from three-species SFSCS measurements on mp-mEYFP-mCherry2-mEGFP hetero-trimers, normalized to the values obtained on cells co-expressing mp-mEGFP, mp-mEYFP and mp-Cherry2.
Author response image 4
Molecular brightness obtained from three-species SFSCS measurements on cells co-expressing mpmCherry2-mEGFP heterodimers and mp-mEYFP, normalized to the values obtained on cells co-expressing mpmEGFP, mp-mEYFP and mp-Cherry2.
Author response image 5
Diffusion times obtained from ACFs in three-species SFSCS measurements on cells expressing mpmEYFP-mCherry2-mEGFP hetero-trimers.
Author response image 6
Relative ACF amplitudes obtained from three-species SFSCS measurements on cells co-expressing mp-mCherry2-mEGFP hetero-dimers and mp-mEYFP.
Only the amplitude ratio for mEGFP and mCherry2 ACFs is shown.
Author response image 7
Diffusion times obtained from ACFs and CCFs in three-species SFSCS measurements on cells coexpressing mp-mCherry2-mEGFP hetero-dimers and mp-mEGFP.
The diffusion times obtained from mEGFP/mCherry2 ACFs and from the CCF are shown here.
Author response image 8
Diffusion dynamics determined in three-species SFSCS measurements on HEK 293T cells coexpressing CD9-mEGFP, LC3-mEYFP, and M2-mCh2 (related to Figure 3D-G).
Author response image 9
Noise analysis of three-species SFSCS measurements described in Figure 3.
Plotted is the SNR (color coded) of ACFs for mEGFP (A), mEYFP (B), and mCherry2 (C) obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mCherry2, as a function of the relative signal to the other two FP species. Date were pooled form two independent experiments in which 31 cells were measured in total.
Author response image 10
Diffusion time (left) and normalized brightness obtained for mEYFP from three-species SFSCS measurements on cells co-expressing mp-mEGFP, mp-mEYFP and mp-mCherry2, pooled in tree groups of data points comprised of the lower, middle and upper third of data points, sorted by SNR.
The brightness was normalized to the average value of all 31 measurements.
Tables
Appendix 1—table 1
Relative cross-correlation (rel.cc.) values (here, same for all channel combinations) for pair-wise or ternary interactions of three-species mixtures.
Values in brackets for pf = 0.7 give rel.cc. values normalized to that of the positive control (i.e., the pair-wise rel.cc. for 1:1 stoichiometry).
| Binding model | pf = 1 | pf = 0.7 |
|---|---|---|
| Pair-wise interactions of dimers (e.g., AA-BB, AA-CC, BB-CC) | 0.50 | 0.41 (0.59) |
| Pair-wise interactions of monomers and homotrimers (e.g., A-B, A-C, B-C, AAA-BBB, AAA-CCC, BBB-CCC) | 0.5 | 0.40 (0.57) |
| Positive control (A-B/A-C/B-C or A-B-C) | 1.0 | 0.7 (1.0) |
| Ternary interactions of dimers (e.g., AA-BB-CC) | 1.0 | 0.83 (1.19) |
| Ternary interactions of monomers and trimers (e.g., A-B-C, AAA-BBB-CCC) | 1.0 | 0.80 (1.14) |
Additional files
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Supplementary file 1
Linker sequences for fluorescent protein (FP) hetero-oligomer constructs and day-to-day variability of molecular brightness values obtained three-species raster spectral image correlation spectrosopcy (RSICS).
(a): Linker sequences of FP hetero-oligomer constructs. (b): Day-to-day variability of molecular brightness values obtained from three-species RSICS measurements.
- https://cdn.elifesciences.org/articles/69687/elife-69687-supp1-v2.docx
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Transparent reporting form
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Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
eLife 10:e69687.
https://doi.org/10.7554/eLife.69687
