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Multiphoton FLIM and spectral imaging of cells and tissues

: König, K.; Riemann, I.; Ehrlich, G.; Ulrich, V.; Fischer, P.


Periasamy, A. ; Society of Photo-Optical Instrumentation Engineers -SPIE-, Bellingham/Wash.:
Multiphoton microscopy in the biomedical sciences IV : 25 - 27 January 2004, San Jose, California, USA
Bellingham, WA: SPIE, 2004 (SPIE Proceedings Series 5323)
ISBN: 0-8194-5231-9
Conference "Multiphoton Microscopy in the Biomedical Sciences" <4, 2004, San Jose, Calif.>
Conference Paper
Fraunhofer IBMT ()

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 (65×62×48 cm(sup 3)) 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 two-photon 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.