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.2013 Aug 20;105(4):848-61.
doi: 10.1016/j.bpj.2013.05.059.

Pulsed interleaved excitation fluctuation imaging

Affiliations

Pulsed interleaved excitation fluctuation imaging

Jelle Hendrix et al. Biophys J..

Abstract

Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.

Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1
PIE-RICS experiments on Venus FP. (A) Logarithmic microtime histograms of all photons detected in the green (top) and red (bottom) detection channel during five images. (Gray hatching) The different PIE channels. (B) The average macrotime image (F = 100) with photons detected in theFGG(t) PIE channel from 100-nM Venus FP freely diffusing in buffer and measured at 5μW excitation power. (C) Typical average spatial ACF of the data in panelB. (D) Three-dimensional representation of the data plotted in panelC. (E) Three-dimensional fit and residual plot for the 100-nM Venus ACF plotted in panelsC andD. (F) Thex- andy-cross sections of the RICS correlation function, fit, and residuals for the data plotted in panelsC andD. (G) ExperimentalG(ξ,0) from RICS (symbols) at 2.5 (blue), 10 (red), 25 (green), and 100μW (pink) excitation power in an aqueous buffer. (Solid line) Fit to Eq. 3. (Plot on theupper panel) Weighted residuals. (Black arrow) Trend with increasing laser power. To allow a direct comparison of their shape, the correlation functions were normalized to the amplitude of the fit function by multiplying the data and fit withN(1-Fb)γ-1. (H andI) The apparent diffusion coefficient (H) and brightness (I) of Venus FP are plotted as a function of laser power. The results from single-point FCS experiments are shown for comparison.
Figure 2
Figure 2
Dual-color PIE-RICS in the cytosol of living HeLa cells. (A) Logarithmic microtime histograms recorded in cells expressing eGFP and mCherry FP at similar concentrations. (Gray hatching) The different PIE channels. (B) Confocal macrotime images of HeLa cells expressing eGFP and mCherry with photons from theFGG(t) (eGFP) andFRR(t) (mCherry) PIE channels. (Open box) Subcellular region where PIE-FI was performed. Various correlation functions are shown for PIE-RICS experiments performed in live cells expressing (C) eGFP and mCherry at similar concentrations, (D) with an excess of eGFP, (E) eGFP-mCherry tandem construct, and (F) mVenus-mCherry tandem construct. Error bars represent the standard deviation and were used for weighted fitting to Eq. 2. In panelsD andF, top views of selected CCFs are shown. The plus-sign in the figure legend indicates the combination of PIE channels into a single macrotime image, while the multiplier sign indicates the cross-correlation calculation of two macrotime images.
Figure 3
Figure 3
Determining the absolute molecular brightness with PIE-FI microscopy. (A) Histogram of the mean intensity per pixel from a series of 100 images of a 20-nM aqueous solution of ATTO488 at 20μW excitation power and 11.11μs pixel dwell time. (B) The photon counting histogram of absolute pixel counts for the same image series analyzed in panelA. (Gray histograms) Raw (not dead-time-corrected) data. (Black histograms) Same data corrected for a 100-ns detection dead time as given by Eq. 13. (C) The relationship between the measured (IM) and corrected (IC) photon counts is plotted assuming a pixel dwell time of 11.11μs for different detection dead times. Thex axis is displayed in counts per pixel dwell time (lower axis) and in megacounts per second (upper axis). (D) The experimental dependence of the measured brightness of ATTO488 determined from samples of various concentration (and hence count rate) assuming different detection dead-time corrections. (E) Dead-time-corrected brightness analysis of ATTO488 and Venus FP in a 10-cP buffer. (F) Molecular brightness measurements of eGFP and eGFP oligomers in live cells. (Left bar chart) Dependence of the apparent monomeric eGFP brightness on laser power and pixel dwell time. (Right bar chart) Molecular brightness for different eGFP tandem constructs ranging from monomers to pentamers.
Figure 4
Figure 4
Measuring picomolar concentrations with RLICS. (A) Experimental microtime histograms of a 100-nM (top), 195-pM (middle), and 6-pM (bottom) ATTO488 solution. (Shaded hatching) Nongated and gatedFGG(t) PIE channels. At 6 pM, they scale was expanded 10 times from 6 ns onward to show the fluorescence. (B) Theoretical versus RICS-measured concentration of a dilution series of ATTO488 from 100 nM to 6 pM in twofold dilution steps with photons from the respective hatched PIE channels in panelA. For reference, the experimentalGGG(0,0) value from the RICS experiment determined by fitting the ACF to Eq. 2 is given on the righty axis. (C) (top) Experimental lifetime weighting filters calculated from the data in theFGG(t) PIE channel (bottom). (D) Theoretical versus measured concentration for the same dilution series as in panelB, when lifetime weighting is used. The nonweighted data and experimentalGGG(0,0) from the RICS experiment are given for comparison.
Figure 5
Figure 5
Dual-fluorophore pseudo-FLIM and dual-color RLICS analysis. Fluorescence lifetime analysis of image data for (A) eGFP and eGFP-mCherry, (B) mVenus and mVenus-mCherry, and (C) mCherry. (D) Calculated idealized fluorescence decay of FRET-quenched (1.17 ns) and unquenched (2.59 ns) eGFP in an eGFP-mCherry tandem convoluted with the experimental IRF. (E) Lifetime weighting filters calculated from the data in panelD. (F) Unfiltered (triangles), FRET-species filtered (circles), and non-FRET species filtered (squares)GGG×RR(ξ,0) of the eGFP-mCherry tandem measured in live cells.
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References

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