An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.
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 (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
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