Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

0492062 - BFÚ 2019 RIV GB eng J - Článek v odborném periodiku
Glazachev, Y.I. - Orlova, D. Y. - Řezníčková, Petra - Bártová, Eva
Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy.
Physical Biology. Roč. 15, č. 3 (2018), č. článku 036008. ISSN 1478-3967. E-ISSN 1478-3975
Grant CEP: GA ČR GBP302/12/G157
Institucionální podpora: RVO:68081707
Klíčová slova: proteins * frap * binding
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 1.818, rok: 2018

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1 ss-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.
Trvalý link: http://hdl.handle.net/11104/0285634