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This work describes the time-resolved fluorescence characteristics
of two different photosensitizers in single cells, in detail
mTHPC and 5-ALA induced PPIX, which are currently clinically used
in photodynamic therapy. The fluorescence lifetime of the drugs was
determined in the cells from time-gated spectra as well as single photon
counting, using a picosecond pulsed diode laser for fluorescence
excitation. The diode laser, which emits pulses at 398 nm with 70 ps
full width at half maximum duration, was coupled to a confocal laser
scanning microscope. For time-resolved spectroscopy a setup consisting
of 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 was used to determine
the fluorescence lifetime from single spots and to record lifetime images.
The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5
ns during incubation from 1 to 6 h. This decrease was probably attributed
to enhanced formation of aggregates during incubation. Fluorescence
lifetime imaging showed that longer lifetimes were correlated
with accumulation in the cytoplasm in the neighborhood of the cell
nucleus, whereas shorter lifetimes were found in the outer cytoplasm.
For cells that were incubated with 5-ALA, a fluorescence lifetime of
7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably
attributed to photoproducts and aggregates of PPIX. In contrast from
fluorescence intensity images alone, different fluorescence species
could not be distinguished. However, in the lifetime image a structured
fluorescence distribution in the cytoplasm was correlated with
the longer lifetime and probably coincides with mitochondria. In conclusion,
picosecond diode lasers coupled to a laser scanning microscope
equipped with appropriate detection units allows time-resolved
spectroscopy and lifetime imaging with high spatial resolution and
provides numerous possibilities in cellular and pharmaceutical research.
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