Abstract
Fluorescence spectroscopy has been a cornerstone of research in membrane dynamics and organization. Technological advances in fluorescence spectroscopy went hand in hand with discovery of various physicochemical properties of membranes at nanometric spatial and microsecond timescales. In this perspective, we discuss the various challenges associated with quantification of physicochemical properties of membranes and how various modes of fluorescence spectroscopy have overcome these challenges to shed light on the structure and organization of membranes. Finally, we discuss newer measurement strategies and data analysis tools to investigate the structure, dynamics, and organization of membranes.
Highlights
The cell membrane, made up of a diverse array of lipids, proteins, and carbohydrates, separates the cytoplasm from the external milieu and is arranged in a bilayer fashion consisting of an outer and an inner leaflet
Fluorescence spectroscopy has been a cornerstone of research in membrane dynamics and organization
Fluorescence techniques that allow determining membrane dynamics and membrane nanoscale organization, as discussed in this review, are very important tools that helped shape our view of cell membranes as it is today
Summary
The cell membrane, made up of a diverse array of lipids, proteins, and carbohydrates, separates the cytoplasm from the external milieu and is arranged in a bilayer fashion consisting of an outer and an inner leaflet. The main tools to investigate organization in a membrane are homo-F€orster resonance energy transfer (homo-FRET), generalized polarization (GP), fluorescence lifetime imaging microscopy (FLIM), FCS diffusion laws, and brightness monitoring techniques. Multiple measurements per observation area are necessary for point FCS based computation of the diffusion law leading, to longer measurement times and possible artifacts caused by photodamage due to repeated excitation. The FCS diffusion law has been extended to analyze complex diffusive modes where the biomolecule under investigation is influenced by domain confinement and influenced by the cytoskeleton.[112] Such an approach will enable one to effectively decouple the relative influences of the cytoskeleton and domain organization on the diffusion of molecules in the cell membrane. GP is typically performed with fluorophores, which exhibit a change in emission maximum depending on the local microenvironment
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