Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes

Abstract

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.

Nonlinear laser imaging of skin lesions

“…The peak in the middle is probably due to photoproducts and photoaggregates of PpIX. In fact, similar results have been found by Kress et al [42] using single-photon fluorescence. We verified that the middle peak is not introduced by the fitting software, by observing similar lifetime distribution even using a double-exponential fitting function.…”

Time-domain fluorescence lifetime imaging applied to biological tissue

“…Recent developments in picosecond pulsed diode lasers, however, offer a considerably cheaper alternative when combined with TCSPC in singlephoton confocal microscopes, e.g. [12] to provide superior 3-D spatial resolution and higher fluorescence signal levels -leading to higher imaging rates.…”

Imaging a photodynamic therapy photosensitizerin vivowith a time-gated fluorescence tomography system

“…[55][56][57][58][59] For example, microscopic TD imaging has been applied for studying photobleaching and lifetime dynamics of PDT photosensitizers in cells. 55,56,60,61 In this paper, we report the application of TD-FDOT for 3-D reconstructions of the fluorescence yield and lifetime of the photosensitizer HPPH in vivo before and after PDT. We first describe a TD-FDOT system, which employs an ultra-short pulse laser, gated imaging, and time-delay techniques.…”