Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate
Tipus de documentArticle
Data publicació2014-01-05
EditorNature
Condicions d'accésAccés obert
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Reconeixement-NoComercial-SenseObraDerivada 3.0 Espanya
Abstract
Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.
ISSN1548-7091
Versió de l'editorhttp://doi.org/10.1038/nmeth.2784
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