Figure 1.

Principles of LS-FCS. (a and b) Intensity images of the plasma membrane adjacent to a glass coverslip in a COS7 cell labeled with DOPE-Atto647N (640 nm excitation at 5 μW). The scale bar represents 10 μm and 1 μm, respectively. (b) The highlighted region (red square in a, 5 μm × 5 μm) is used to select the position of the LS (green line). (c) Kymograph representing the time evolution of the fluorescence intensity along the scanned line. The scale bar represents 1 μm. A total of 2,170,000 lines were sampled for 137 s at 63 μs per line. At full resolution, each line consists of 2,852 nonequally spaced points. (d) A zoomed region of the kymograph reveals the scarcity of detected photons. The scale bar represents 100 nm. (e) Individual photons (stars) are recorded in a TTTR format and the arrival time of each photon is converted into its exact position along the scanned line (blue lines; the black line indicates movement of the scanner). The time and position differences, ∂t and ∂x, respectively, from which spatiotemporal correlation curves are calculated are shown for a selected pair of photons. (f) Spatiotemporal autocorrelation for DOPE-Atto647N in a COS7 cell from the image shown in (a) and (b) at confocal (i.e., diffraction-limited) resolution. The projections along the space and time axes are provided to highlight the shape of the autocorrelation function reflecting the time decay and PSF profile, respectively. To see this figure in color, go online.