Abstract
By recording the full fluorescence spectra and super-resolved positions of ∼106 individual polarity-sensing solvatochromic molecules, we reveal compositional heterogeneity in the membranes of live mammalian cells with single-molecule sensitivity and ∼30 nm spatial resolution. This allowed us to unveil distinct polarity characteristics of the plasma membrane and the membranes of nanoscale intracellular organelles, a result we found to be due to differences in cholesterol levels. Within the plasma membrane, we observed the formation of low-polarity, raft-like nanodomains upon cholesterol addition or cholera-toxin treatment, but found this nanoscale phase separation absent in native cells. The ultimate sensitivity achieved through examining the spectra of individual molecules thus opens the door to functional interrogations of intracellular physicochemical parameters at the nanoscale.
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