Fig. 4.

TIRFM detection of BODIPY-sphingolipids in the plasma membrane of a Clone 15 cell at 37 °C. Stacks of 120 frames were acquired before and after fixation. Images were background subtracted and averaged through the stack to improve signal-to-noise ratio. (A) BODIPY-sphingolipid domains are visible in the plasma membrane of the living Clone 15 cell under physiological conditions. (B and C) Enlarged view of the region outlined in A shows the BODIPY-sphingolipid membrane domains (B) in the living cell and (C) following glutaraldehyde fixation. The sizes and distributions of the sphingolipid domains were unaltered by fixation. The high fluorescence of the cellular microextensions is an artifact of background correction and does not represent a local enrichment of sphingolipids; these microextensions have the same fluorescent intensities as the nondomain regions on the cell in the image without background correction (Fig. S8).