The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.

EMBL Australia Node in Single Molecule Science School of Medical Sciences Sydney New South Wales Australia; ARC Centre of Excellence in Advanced Molecular Imaging University of New South Wales Sydney New South Wales Australia

EMBL Australia Node in Single Molecule Science School of Medical Sciences Sydney New South Wales Australia; ARC Centre of Excellence in Advanced Molecular Imaging University of New South Wales Sydney New South Wales Australia; Allen Institute for Brain Science Seattle Washington

Imaging Methods Core Facility at BIOCEV Faculty of Sciences Charles University Vestec Czech Republic

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