The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of
small molecules and, hence, controlling the rates of intra-cellular reactions. There is significant
interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of
fluorescence lifetime imaging microscopy (FLIM) of the molecular rotor BODIPY C10 in the
membranes of live Escherichia coli (E. coli) bacteria to permit direct quantification of the viscosity.
Using this approach we investigated the viscosity in live E. coli cells, spheroplasts and liposomes
made from E. coli membrane extracts. For live cells and spheroplasts the viscosity was measured at
both room temperature (23o C) and the E. coli growth temperature (37o C), while the membrane
extract liposomes were studied over a range of measurement temperatures (5-40o C). At 37o C we
recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that
previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus
vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering
within their liquid-phase plasma membranes.