1 March 2008 Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation
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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.
© (2008) Society of Photo-Optical Instrumentation Engineers (SPIE)
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). https://doi.org/10.1117/1.2907316 . Submission:
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