A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in “spot scan” mode. In addition, a time-correlated single photon counting module (TCSPC) was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping).To prove and calibrate the system, the time-resolved fluorescence characteristics of the mitochondrial marker Rhodamine 123 and 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated in solution and in cell culture. Different lifetimes could be found in different cell compartiments. During illumination, the lifetimes decreased significantly. From photobleaching experiments different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In conclusion FLIM, using ps diode lasers and TCSPC techniques is a valuable method to selectively identify and localize various metabolites of fluorescent probes during laser scanning microscopy.

© 2003 SPIE

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