Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 5, Pages -Publisher
MDPI
DOI: 10.3390/ijms23052482
Keywords
stimulated emission depletion (STED); fluorescent protein; super-resolution; ultrafast spectroscopy; excited state absorption (ESA); Vimentin
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By combining steady-state and ultrafast spectroscopy with gated STED imaging, this study demonstrates that F99S/M153T/V163A GFP is an efficient genetic reporter for STED imaging, with no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.
In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.
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