Figure 1

Analysis of sterol mobility by raster image correlation spectroscopy (RICS). CHO cells were pulse-labeled with BChol followed by a chase to obtain the steady-state distribution. The cells were placed on a temperature-controlled stage of a home-built 2P-microscope maintained at 35 ± 1°C, and images were acquired without pause at a frame rate of 0.52 s. Three areas of a cell incubated with BChol were analyzed with raster image correlation spectroscopy (RICS), panel A. The areas were selected to include the membrane (box 1), the cytoplasm with no apparent BChol vesicles (box 2), and an area close the ERC (box3). Inlet 1–3 show fits of the Brownian diffusion model to the RICS autocorrelation function in these areas and the residuals of the fit. The analysis showed that diffusion near the ERC was a factor of two slower than in the membrane. Nevertheless, the molecules in the central part of the cell were still mobile. This is illustrated in panels B-D (scale bar = 5 μm), which show the correlation between the cell at time t = 0 (colored red) with images at time t = 0.52, 5.2 and 10.4 s, respectively (colored green). The intracellular dynamics is especially apparent in panel B’-D’ showing the dynamics in the ROI in panels B-D.