Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation.
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.
Lu
Sijia
S
Fudan University, Department of Physics, 200433, Shanghai, China.
Chen
Ji-Yao
JY
Zhang
Yu
Y
Ma
Jiong
J
Wang
Pei-Nan
PN
Peng
Qian
Q
D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D011524:Protoporphyrins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D011524:Protoporphyrins; C028025:protoporphyrin IX