The functions of cell plasma membranes strongly rely on their physicochemical properties, which determine the membrane structure, control the transport of molecular species, and determine the folding and function of membrane proteins. Among these properties, the membrane electrostatics, phase state, hydration, and dynamics are of particular importance and their monitoring appears critical to further understand the structure and functions of biomembranes. Among the different techniques used to reach this aim, fluorescence-based techniques are of special interest, due notably, to their single-molecule sensitivity and their ability to operate from the molecular to the animal level. However, several intrinsic properties of the membranes render their analysis by fluorescence techniques particularly challenging. Indeed, the membrane is highly anisotropic with steep gradients of its physico-chemical properties within lipid membrane width, which does not exceed 5 nm. In addition, the membrane interior is viscous and constrained, which strongly limits the probe motion and relaxation processes. As a result, approximating the probe environment as an isotropic medium is no more valid and thus, a switch to a molecular-scale interpretation of the fluorescence data is needed. To reach this aim, probes with controlled and precise depth and orientation in the bilayer should be designed. In addition, since multiple parameters are to be measured, multiparametric probes able to simultaneously monitor several parameters through different channels should be developed.
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