Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan

Herbert Schneckenburger; Karl Stock; Joerg Eickholz; Wolfgang S. L. Strauss; Marco Lyttek; Reinhard Sailer

doi:10.1117/12.410626

18 December 2000 Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan

Herbert Schneckenburger, Karl Stock, Joerg Eickholz, Wolfgang S. L. Strauss, Marco Lyttek, Reinhard Sailer

Author Affiliations +

Proceedings Volume 4164, Laser Microscopy; (2000) https://doi.org/10.1117/12.410626
Event: EOS/SPIE European Biomedical Optics Week, 2000, Amsterdam, Netherlands

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 I_F 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.

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Herbert Schneckenburger, Karl Stock, Joerg Eickholz, Wolfgang S. L. Strauss, Marco Lyttek, and Reinhard Sailer "Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan", Proc. SPIE 4164, Laser Microscopy, (18 December 2000); https://doi.org/10.1117/12.410626

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