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Fluorescence and polarization imaging of membrane dynamics in living cells
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Paper Abstract

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.

Paper Details

Date Published: 12 February 2009
PDF: 8 pages
Proc. SPIE 7176, Dynamics and Fluctuations in Biomedical Photonics VI, 717607 (12 February 2009); doi: 10.1117/12.808788
Show Author Affiliations
M. Wagner, Hochschule Aalen (Germany)
P. Weber, Hochschule Aalen (Germany)
T. Bruns, Hochschule Aalen (Germany)
W. S. L. Strauss, Univ. Ulm (Germany)
H. Schneckenburger, Hochschule Aalen (Germany)
Univ. Ulm (Germany)

Published in SPIE Proceedings Vol. 7176:
Dynamics and Fluctuations in Biomedical Photonics VI
Valery Viktorovich Tuchin; Lihong V. Wang; Donald D. Duncan, Editor(s)

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