Research Papers

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

[+] Author Affiliations
Matthias Kress, Thomas Meier, Rudolf Steiner, Frank Dolp

Institute for Laser Technologies in Medicine and Metrology, Helmholtzstr. 12, 89081 Ulm, Germany

Rainer Erdmann, Uwe Ortmann

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Angelika Ru¨ck

Institute for Laser Technologies in Medicine and Metrology, Helmholtzstr. 12, 89081 Ulm, Germany

J. Biomed. Opt. 8(1), 26-32 (Jan 01, 2003). doi:10.1117/1.1528595
History: Received Nov. 8, 2001; Revised Apr. 16, 2002; Revised Aug. 14, 2002; Accepted Aug. 30, 2002; Online January 14, 2003
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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. © 2003 Society of Photo-Optical Instrumentation Engineers.

© 2003 Society of Photo-Optical Instrumentation Engineers

Citation

Matthias Kress ; Thomas Meier ; Rudolf Steiner ; Frank Dolp ; Rainer Erdmann, et al.
"Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes", J. Biomed. Opt. 8(1), 26-32 (Jan 01, 2003). ; http://dx.doi.org/10.1117/1.1528595


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