Measuring membrane fluidity in Bacillus subtilis using TIR-FCS

Overview & Synopsis

Bacteria need to adapt their membrane fluidity in response to a wide range of stimuli, whether physical or chemical. For instance, the bacterium Bacillus subtilis changes the fatty acid composition of its membrane in response to a change in temperature. Changes in membrane fluidity were either estimated from biochemical methods like fatty acids analysis or indirectly assessed using environment-sensitive probes, but never directly measured. We show here that Fluorescence Correlation Spectroscopy (FCS) used on a TIRF microscope (TIR-FCS) can be used to measure the diffusion of a well-known membrane marker, Nile Red. Because relative changes in diffusion coefficients are directly linked to a change in membrane fluidity, we can quantify changes in membrane fluidity that could so far only be qualitatively assessed. To facilitate the analysis of the large amount of data generated by TIR-FCS, we developed a simple method to automatically exclude artefactual FCS curves. As a result, we could perform high-throughput FCS measurements revealing the impact of changes in temperature and of sporulation on membrane fluidity.