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Axially resolved polarisation microscopy of membrane dynamics in living cells
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Paper Abstract

Membrane dynamics has a large impact on cellular uptake and release of various metabolites or pharmaceutical agents. For a deeper understanding of the cellular processes involved, we used U373-MG human glioblastoma cells as a model system. As conventional microscopy does not permit to investigate individual layers in living cells, we used structured illumination techniques and total internal reflection fluorescence microscopy (TIRFM) to analyse the plasma membrane and intracellular membranes of living cells selectively. Optical image sections provide a high resolution and the possibility of 3D reconstruction. Membranes of living cells were characterized by the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). Due to its spectral and kinetic properties this fluorescence marker appears appropriate for measuring membrane stiffness and fluidity. After excitation with linearly polarized laser pulses, membrane fluidity of human glioblastoma cells was determined by measurements of steady-state and time-resolved fluorescence anisotropy r(t), since with increasing viscosity of the environment, the rotation of an excited molecule is impeded. The corresponding time constant &tgr;r of molecular relaxation decreased with temperature and increased with the amount of cholesterol. In addition, fluorescence anisotropy r(t) values of the plasma membrane were larger than the values of intracellular membranes for all temperatures in the range of 16°C≤T≤41°C.

Paper Details

Date Published: 13 July 2007
PDF: 7 pages
Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 663309 (13 July 2007); doi: 10.1117/12.727780
Show Author Affiliations
Michael Wagner, Hochschule Aalen (Germany)
Petra Weber, Hochschule Aalen (Germany)
Herbert Schneckenburger, Hochschule Aalen (Germany)
Univ. Ulm (Germany)

Published in SPIE Proceedings Vol. 6633:
Biophotonics 2007: Optics in Life Science
Jürgen Popp; Gert von Bally, Editor(s)

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