1 March 2008 Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation
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J. of Biomedical Optics, 13(2), 024014 (2008). doi:10.1117/1.2907316
Abstract
Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.
Sijia Lu, Ji Yao Chen, Yu Zhang, Jiong Ma, Pei Nan Wang, Qian Peng, "Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation," Journal of Biomedical Optics 13(2), 024014 (1 March 2008). http://dx.doi.org/10.1117/1.2907316
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KEYWORDS
Luminescence

Fluorescence spectroscopy

Photodynamic therapy

Confocal microscopy

Signal detection

Femtosecond phenomena

Digital image correlation

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