Paper
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
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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.
© (2000) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
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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Cited by 3 scholarly publications.
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KEYWORDS
Luminescence

Temperature metrology

Fluorescence spectroscopy

Plasma

Pulsed laser operation

Reflection

Polarization

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