Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques

Claudia Scalfi-Happ; Frank Dolp; Florian Forster; Angelika Rueck

doi:10.1117/12.545472

10 September 2004 Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques

Claudia Scalfi-Happ, Frank Dolp, Florian Forster, Angelika Rueck

Author Affiliations +

Proceedings Volume 5462, Biophotonics Micro- and Nano-Imaging; (2004) https://doi.org/10.1117/12.545472
Event: Photonics Europe, 2004, Strasbourg, France

Abstract

A time-correlated single photon counting (TCSPC) module (SPC-730, Becker & Hickl, Germany) was connected to a laser scanning microscope (Zeiss, Germany) equipped with an ultrafast photomultiplier. Short pulse excitation was achieved with two laser diodes emitting at 398nm and 434nm with a pulse duration of 70ps and 60 ps (PicoQuant, Germany) to allow intracellular fluorescence lifetime imaging (FLIM). With this setup, fluorescence lifetime of the mitochondrial marker Rhodamine 123 could be studied in solution under the same instrumental conditions as used for fluorescence lifetime imaging of cell monolayers. With the same set of parameters, fluorescence lifetime of Rhodamine 123 was calculated with good reproducibility in mitochondria of living cells. We present here a comparison of different fitting routines, including a multiexponential fitting based on the method of Laplace transformation. Fluorescence lifetimes calculated with the multiexponential fitting routine proved to be particularly useful to study the distribution of 5-ALA metabolites in cell monolayers.

Citation Download Citation

Claudia Scalfi-Happ, Frank Dolp, Florian Forster, and Angelika Rueck "Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques", Proc. SPIE 5462, Biophotonics Micro- and Nano-Imaging, (10 September 2004); https://doi.org/10.1117/12.545472

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