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Proceedings Paper

Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan
Author(s): Herbert Schneckenburger; Karl Stock; Joerg Eickholz; Wolfgang S. L. Strauss; Marco Lyttek; Reinhard Sailer
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Paper Abstract

A compact device for variable-angle total internal reflection flourescent microscopy was developed. A pulsed Nd:YAG laser operated at 355 nm was adapted using a multimode quartz fiber and collimating optics with a variable angle of incidence between 64 degrees and 72 degrees. Fluorescence spectra of BKEz-7 endothelial cells incubated with the membrane marker 6-dodecanoyl-2- dimethylamino-naphthalene were measured under TIR illumination as a function of the angle of incidence, incubation time and temperature. Emission bands around 440 nm and 490 nm were detected corresponding to laurdan locate within the gel phase or liquid crystalline phase of cellular lipids, respectively. The generalized polarization GP = (I440-I490)/(I440+I490) was used as a measure of intracellular temperature with a precision of ±1°C in the physiologically interesting range between 35°C and 38°C. Following pulsed laser excitation, the time delay between excitation and fluorescence detection was varied. A time gate at 10-15 ns after laser excitation revealed to be an optimum for spectral discrimination of the two emission bands. Fluorescence intensity IF of both bands decreased continuously when the angle of incidence Θ was increased. Between Θ = 68° and 72° the angular dependence corresponded to a fluorophore located within a thin layer (plasma membrane) at 150-200nm distance from the light reflecting surface. Between Θ=65° and 68° additional contributions from intracellular membranes were observed.

Paper Details

Date Published: 18 December 2000
PDF: 7 pages
Proc. SPIE 4164, Laser Microscopy, (18 December 2000); doi: 10.1117/12.410626
Show Author Affiliations
Herbert Schneckenburger, Institut fuer Angewandte Forschung and ILMM Univ. Ulm (Germany)
Karl Stock, Institut fuer Angewandte Forschung and Univ. Ulm (Germany)
Joerg Eickholz, Institut fuer Angewandte Forschung (Germany)
Wolfgang S. L. Strauss, ILMM Univ. Ulm (Germany)
Marco Lyttek, Institut fuer Angewandte Forschung (Germany)
Reinhard Sailer, ILMM Univ. Ulm (Germany)

Published in SPIE Proceedings Vol. 4164:
Laser Microscopy
Karsten Koenig; Hans J. Tanke; Herbert Schneckenburger, Editor(s)

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