Fluorescence and polarization imaging of membrane dynamics in living cells
2009
Methods of wide field fluorescence microscopy for measuring membrane dynamics in living cells are described. These
methods are based on laser pulse excitation of the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene
(laurdan) whose emission spectra, fluorescence decay kinetics and anisotropies are sensitive to membrane stiffness and
fluidity. Plasma membranes are selected by illumination with an evanescent electromagnetic field and distinguished from
intracellular membranes assessed by whole cell illumination. While fluorescence spectra of laurdan appeared red-shifted
with decreasing membrane stiffness, fluorescence anisotropy and rotational relaxation times were reduced with
increasing membrane fluidity. Membrane stiffness was found to increase with decreasing temperature and increasing
amounts of cholesterol. In addition, membrane stiffness of the plasma membrane was always higher than that of
intracellular membranes. These effects may have some influence on pathogenesis of certain diseases, uptake of
pharmaceutical agents or cell aging. Present experiments are limited to fluorescence microscopy with total internal
reflection (TIR) or epi-illumination, but corresponding methods can also be used for screening of larger cell collectives,
e.g. in microtiter plates.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
15
References
0
Citations
NaN
KQI