Multiphoton FLIM and spectral imaging of cells and tissues

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.