Lifetime-based photoconversion of EGFP as a tool for FLIM
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
30394285
DOI
10.1016/j.bbagen.2018.10.016
PII: S0304-4165(18)30337-4
Knihovny.cz E-resources
- Keywords
- Fluorescence lifetime imaging, Leptomycin B, Nucleophosmin, Photobleaching, Phototransformation, Protein tracking,
- MeSH
- Cell Adhesion MeSH
- Fluorescence MeSH
- Microscopy, Fluorescence methods MeSH
- Photochemistry methods MeSH
- HEK293 Cells MeSH
- HeLa Cells MeSH
- Nuclear Proteins chemistry MeSH
- Kinetics MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Mutation MeSH
- Fatty Acids, Unsaturated chemistry MeSH
- Nucleophosmin MeSH
- Solvents chemistry MeSH
- Viscosity MeSH
- Green Fluorescent Proteins chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- enhanced green fluorescent protein MeSH Browser
- Nuclear Proteins MeSH
- leptomycin B MeSH Browser
- Fatty Acids, Unsaturated MeSH
- NPM1 protein, human MeSH Browser
- Nucleophosmin MeSH
- Solvents MeSH
- Green Fluorescent Proteins MeSH
BACKGROUND: EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS: Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT: Our data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS: Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. GENERAL SIGNIFICANCE: The reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.
References provided by Crossref.org
Interferometric excitation fluorescence lifetime imaging microscopy
NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization
