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Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity
One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599333/ https://www.ncbi.nlm.nih.gov/pubmed/33128009 http://dx.doi.org/10.1038/s42003-020-01316-2 |
Sumario: | One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown how many of the proposedly fluorescent molecules of a sample are indeed fluorescent (for example due to incomplete maturation, or the presence of photophysical dark states). Here, we use a plasmonic nanocavity-based method to measure absolute quantum yield values of commonly used fluorescent proteins. The method is calibration-free, does not require knowledge about maturation or potential dark states, and works on minute amounts of sample. The insensitivity of the nanocavity-based method to the presence of non-luminescent species allowed us to measure precisely the quantum yield of photo-switchable proteins in their on-state and to analyze the origin of the residual fluorescence of protein ensembles switched to the dark state. |
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