Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.
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.