Studying Membrane Properties Using Fluorescence Lifetime Imaging Microscopy (FLIM)

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to investigate the structure and composition of biological membranes. A wide variety of fluorescent probes suitable for FLIM experiments have been described. These compounds differ strongly in the details of their incorporation into membranes and in their responses toward changes in the membrane composition. In this chapter, we discuss and compare different classes of fluorescent membranes probes and their applications to studying biological membranes. We devote a section to a detailed description of fluorescent molecular rotors and their application to measuring local viscosity. As Fo ̈rster resonance energy transfer (FRET) can be directly measured by changes in the donor fluorescence lifetime, FLIM is a very robust method to deter- mine the distances between FRET pairs or the local concentrations of FRET-based membrane probes. Thus, we also discuss advantages and challenges of FRET-FLIM in the context of biological membranes. As biological membranes are considerably dynamic systems, imaging speed is often the limiting factor in biological FLIM experiments. Thus, novel fast imaging approaches and analysis methods to alleviate the issue of low photon statistics are also presented