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 …
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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 …
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 …
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). …
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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 …
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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 …
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 …
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 …
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 …
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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 …
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 …
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 …
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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 …
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 …
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 …
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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 …
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, …
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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 …
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 …
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 …
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 …
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
- https://cdn.elifesciences.org/articles/69687/elife-69687-transrepform1-v2.pdf
