Time-resolved fluorescence imaging of photosensitiser distributions in mammalian cells using a picosecond laser line-scanning microscope

A fluorescence microscope using line-excitation generated from a pulsed pic osecond dye laser source has been combined with a gated sub-nanosecond high repetition rate (800 kHz) image intensifier to enable time-gated fluoresce nce lifetime imaging. Using this system, several potential photosensitisers for photodynamic therapy (PDT) have been studied. The fluorescence of the anionic disulphonated aluminium phthalocyanine (AlPcS2) and cationic pyridi niumzinc(II) phthalocyanine (ZnPPC) has been imaged in hamster V79-4 fibrob lasts and compared with AlPcS2 and meta-tetra(hydroxyphenyl) chlorin (m-THP C) in murine macrophages (J774A.1). Lifetime images were obtained from nano second delay sequences of fluorescence images with detector gate widths of 700 ps by fitting the data to single lifetime decays. AlPcS2 and ZnPPC fluo rescence appeared to be localised predominantly in perinuclear sites and ab sent from the nucleus. Similar regional average lifetime ranges in both cel l lines for AlPcS2 were observed, varying between 4 and 5 ns at the most in tense sites of fluorescence. ZnPPC exhibited more uniform lifetime maps wit h average lifetimes of 2.5-3.0 ns. The chlorin m-THPC fluorescence was also extranuclear and appeared more homogeneously distributed, but was strongly affected by photobleaching. This study demonstrates the utility of the las er line-scanning technique for rapid acquisition of time-gated fluorescence lifetime images. (C) 2001 Elsevier Science B.V. All rights reserved.