Mr. Gunter Ehrlich Profile

Gunter Ehrlich

at Friedrich-Schiller-Univ Jena

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Proceedings Article | 21 June 2004 Paper

Multiphoton FLIM and spectral imaging of cells and tissues

Karsten Konig, Iris Riemann, Gunter Ehrlich, Volker Ulrich, Peter Fischer

Proceedings Volume 5323, (2004) https://doi.org/10.1117/12.528499

KEYWORDS: Luminescence, Fluorescence lifetime imaging, Fluorescence resonance energy transfer, Imaging spectroscopy, Microscopes, Picosecond phenomena, Tissues, Molecules, Femtosecond phenomena, Fluorescent proteins

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Five-dimensional (5D) multiphoton measurements with submicron spatial resolution, 270 ps temporal resolution and 5 nm spectral resolution have been performed on living cells and tissues at 750 nm - 850 nm laser excitation. A compact (65x62x48 cm³) multiport laser scanning microscope TauMap (JenLab GmbH) equipped with fast PMT and CCD camera, SPC 830 time-correlated single photon counting board and Sagnac interferometer was used. Laser excitation radiation was provided by a tuneable MaiTai Ti:sapphire femtosecond laser as well as by a 405 nm 50 MHz picosecond laser diode. The spectral and temporal fluorescence behaviour of intratissue chloroplasts of water plant leafs, of a variety of exogenous fluorophores as well as of fluorescent proteins in transfected brain cells have been studied. When calculating fluorescence lifetime images (FLIM) we found differences in intracellular twophoton fluorescence lifetimes vs. one-photon fluorescence lifetimes. Multiphoton FLIM-FRET and multiphoton spectral FRET studies have been performed in living HBMEC brain cells using CFP and YFP fusion proteins. It was shown that FLIM-FRET data depend on laser power due to photodestructive multiphoton effects. This has to be considered in long-term fluorescence resonance energy transfer studies of dynamic protein-protein interactions.

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