Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

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Abstract
The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of ∼50 nm, using ‘single dye tracing’. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid‐specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid‐ordered phase: the probe showed confined but fast diffusion, with high partitioning (∼100‐fold) and long residence time (∼13 s). The analogous probe with mono‐unsaturated chains diffused predominantly unconfined within the membrane. With ∼15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 μm (0.2–2 μm), covering ∼13% of total membrane area. Both the liquid‐ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent‐resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.