Analysis of sterol mobility by temporal image correlation spectroscopy (TICS). Cells were labeled and imaged as described in the legend to Figure 1. The intracellular transport of BChol was examined by temporal image correlation spectroscopy (TICS) and number and brightness analysis (N&B). Panel A shows a map of diffusion constants (D) in the cell ranging from ~1.5 – 0 μm2/s. Panels B-D show the average fluorescence intensity, the apparent number of molecules (N) and the apparent brightness (B), respectively. From a comparison of the average intensity and the apparent number of molecules we infer that the majority of BChol is found in the ERC. Although, the brightness map is relatively uniform (notice that the white areas are outside the cell) we still see signs of aggregation. This is most likely due to BChol binding transiently to intracellular structures (see text for discussion). Finally, Panel E shows the relationship between N, B and the diffusion constant of BChol. By comparison of N and D, it can be seen that the diffusion constant is inversely dependent on the number of molecules. As the brightness is constant over the ROI, we conclude that the decrease in diffusion constant is caused by molecular crowding rather than by aggregation of BChol.