On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

Abstract

Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.

Figure 1

Illustration of a simultaneous FRAP and ITIR-FCS experiment performed on living CHO cells expressing GFP-GPI. The particle number (N) and diffusion coefficient (D) maps are given in (A) for the three substacks (prebleach, recovery, and postrecovery). The dimensions of the cell images are 25.6 × 6.4 μm. A color-scale bar of N and D is next to the maps. (B) Autocorrelation functions (gray curves) for each observation area (2 × 2 binned pixels, 0.35 μm²) in each of the three substacks. The best fits of the correlation curves are also shown (black lines) together with the average values of D obtained from all the fits. The whole intensity trace in the FRAP bleach region (a circle of 3.2-μm radius) is plotted in (C) and the definition of the prebleach, recovery, and postrecovery are schematically depicted. The recovery trace corrected for bleaching by reference area correction is shown in (D) (gray line) together with a fit by Eq. 2 (black line) and the fitted value of D. To see this figure in color, go online.

Figure 2

Comparison of D_FCS with D_FRAP for simulations (A) and living CHO cells (B). (A) Comparison of D_FCS with D_FRAP values obtained for simulated stacks using the three bleach correction strategies. The horizontal and vertical lines correspond to the actual values of D used in the simulations. The dotted line corresponds to D_FRAP = D to facilitate comparison. (B) Comparison of D_FCS and D_FRAP values obtained for living CHO cells expressing GFP-GPI. All recovery traces were bleach-corrected using reference area correction. (Solid symbols) Results from one individual cell each. (Open square) Average D_FCS and D_FRAP with respective standard deviations. Note that for one of the cells (black circle) both the D_FRAP and D_FCS are considerably larger than the average of the rest of the cells; this cell was excluded from calculating the average displayed by the open square. Dotted line corresponds to D_FCS = D_FRAP to facilitate comparison.

Figure 3

An example of the influence of the selection of the reference area on bleach correction by the reference area strategy. The two reference areas are schematically depicted in (A) superimposed on the image of a CHO cell expressing GFP-GPI (average projection of 100 frames immediately following the end of the FRAP bleaching pulse). The dimensions of the cell image are 25.6 × 6.4 μm. The average intensity traces from each of the reference areas are plotted in (B) (for the frames starting with the first frame after the FRAP bleaching pulse and ending with the last frame of the stack). The recovery trace was corrected by the two individual reference traces and by the average reference trace (the average intensity trace of both areas) (C). The best fits of the traces with Eq. 2 are also plotted and the fitted values of D are given in the figure legend.