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Background: We wanted to investigate the physical state of biological membranes in live cells under the most physiological conditions possible. Methods: For this we have been using laurdan, C-laurdan or M-laurdan to label a variety of cells, and a biphoton microscope equipped with both a thermostatic chamber and a spectral analyser. We also used a flow cytometer to quantify the 450/530 nm ratio of fluorescence emissions by whole cells. Results: We find that using all the information provided by spectral analysis to perform spectral decomposition dramatically improves the imaging resolution compared to using just two channels, as commonly used to calculate generalized polarisation (GP). Coupled to a new plugin called Fraction Mapper, developed to represent the fraction of light intensity in the first component in a stack of two images, we obtain very clear pictures of both the intra-cellular distribution of the probes, and the polarity of the cellular environments where the lipid probes are localised. Our results lead us to conclude that, in live cells kept at 37°C, laurdan, and M-laurdan to a lesser extent, have a strong tendency to accumulate in the very apolar environment of intra-cytoplasmic lipid droplets, but label the plasma membrane (PM) of mammalian cells ineffectively. On the other hand, C-laurdan labels the PM very quickly and effectively, and does not detectably accumulate in lipid droplets. Conclusions: From using these probes on a variety of mammalian cell lines, as well as on cells from Drosophila and Dictyostelium discoideum, we conclude that, apart from the lipid droplets, which are very apolar, probes in intracellular membranes reveal a relatively polar and hydrated environment, suggesting a very marked dominance of liquid disordered states. PMs, on the other hand, are much more apolar, suggesting a strong dominance of liquid ordered state, which fits with their high sterol contents.

Using spectral decomposition of the signals from laurdan-derived probes to evaluate the physical state of membranes in live cells