Applications of fluorescence lifetime spectroscopy and imaging to lipid domains in vivo.

Lipid domains are part of the current description of cell membranes and their involvement in many fundamental cellular processes is currently acknowledged. However, their study in living cells is still a challenge. Fluorescence lifetimes have and will continue to play an important role in unraveling the properties and function of lipid domains, and their use in vivo is expected to increase in the near future, since their extreme sensitivity to the physical properties of the membrane and the possibility of optical imaging are particularly suited to deal with the hurdles that are met by researchers. In this review, a practical guide on the use of fluorescence lifetimes for the study of this subject is given. A section is devoted to studies in vitro, particularly membrane model systems, and how they are used to better design and correctly interpret results obtained in living cells. Criteria are presented for selecting suitable probes to solve each problem, drawing attention to factors sometimes overlooked and which may affect the fluorescence lifetime such as subcellular distribution and concentration of the probe. The principal groups of lifetime probes for lipid domains: (i) fluorescent lipid analogues, (ii) other lipophilic probes, and (iii) fluorescent proteins, and respective applications are briefly described and lab tips about the labeling of living cells are provided. The advantages and complementarities of spectroscopy (cuvette) work and fluorescence lifetime imaging microscopy are presented and illustrated with three selected case studies: (i) the finding of a new type of lipid rafts in yeast cells; (ii) the detection of liquid ordered type heterogeneity in animal cells below optical resolution; and (iii) establish a role for the transmembrane domain of influenza virus hemagglutinin with cholesterol-enriched domains in mammalian cells.