Tags

Type your tag names separated by a space and hit enter

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate.
Nat Methods 2014; 11(2):156-62NM

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.

Authors+Show Affiliations

ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24390439

Citation

Durisic, Nela, et al. "Single-molecule Evaluation of Fluorescent Protein Photoactivation Efficiency Using an in Vivo Nanotemplate." Nature Methods, vol. 11, no. 2, 2014, pp. 156-62.
Durisic N, Laparra-Cuervo L, Sandoval-Álvarez A, et al. Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods. 2014;11(2):156-62.
Durisic, N., Laparra-Cuervo, L., Sandoval-Álvarez, A., Borbely, J. S., & Lakadamyali, M. (2014). Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nature Methods, 11(2), pp. 156-62. doi:10.1038/nmeth.2784.
Durisic N, et al. Single-molecule Evaluation of Fluorescent Protein Photoactivation Efficiency Using an in Vivo Nanotemplate. Nat Methods. 2014;11(2):156-62. PubMed PMID: 24390439.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. AU - Durisic,Nela, AU - Laparra-Cuervo,Lara, AU - Sandoval-Álvarez,Angel, AU - Borbely,Joseph Steven, AU - Lakadamyali,Melike, Y1 - 2014/01/05/ PY - 2013/04/29/received PY - 2013/11/22/accepted PY - 2014/1/7/entrez PY - 2014/1/7/pubmed PY - 2014/3/26/medline SP - 156 EP - 62 JF - Nature methods JO - Nat. Methods VL - 11 IS - 2 N2 - 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. SN - 1548-7105 UR - https://www.unboundmedicine.com/medline/citation/24390439/Single_molecule_evaluation_of_fluorescent_protein_photoactivation_efficiency_using_an_in_vivo_nanotemplate_ L2 - http://dx.doi.org/10.1038/nmeth.2784 DB - PRIME DP - Unbound Medicine ER -