title: High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy.
abstract: Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.
authors.affiliation: Key Laboratory of Biomedical Photonics, Ministry of Education of China, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Tong-Sheng
authors.initials: TS
authors.lastname: Chen
authors.forename: Shao-Qun
authors.initials: SQ
authors.lastname: Zeng
authors.forename: Qing-Ming
authors.initials: QM
authors.lastname: Luo
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
mesh_terms: D003952:Diagnostic Imaging; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D017785:Photons
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins