Microspectrofluorometry and Polarisation Microscopy of Membrane Dynamics in Living Cells.

Organization and dynamics of cell membranes have a large impact on cellular uptake and release of various metabolites or pharmaceutical agents. Membranes of living cells are characterized using the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan). Membranes are assessed by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry and total internal reflection fluorescence microscopy (TIRFM). Previously Membrane stiffness was related to spectral properties and expressed by the generalized polarization (GP), which depends on the phase of membrane lipids. GP generally decreased with temperature, and was always higher for the plasma membrane than for intracellular membranes. In addition, membrane fluidity was assessed by measurements of steady-state and time-resolved fluorescence anisotropy r(t), since with increasing viscosity, i. e. decreasing fluidity of the environment, the rotation of an excited molecule is impeded. The parallel and perpendicular components of the fluorescence emission from the sample were measured simultaneously using an imaging device with polarization sensitivity. In addition to GP, fluorescence anisotropy r(t) proved to be an independent measure for characterizing membrane dynamics. So far, membrane dynamics depended on temperature, growth phase as well as the on the intracellular amount of cholesterol. [ABSTRACT FROM AUTHOR]