| Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). | |
| | |
MedLine Citation:
|
PMID: 23045631 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over- and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2. |
| | |
Authors:
|
Sang-Hyuk Lee; Jae Yen Shin; Antony Lee; Carlos Bustamante |
Publication Detail:
|
Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. Date: 2012-10-08 |
Journal Detail:
|
Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 109 ISSN: 1091-6490 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2012 Oct |
Date Detail:
|
Created Date: 2012-10-24 Completed Date: 2013-01-07 Revised Date: 2013-02-19 |
Medline Journal Info:
|
Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
|
Languages: eng Pagination: 17436-41 Citation Subset: IM |
Affiliation:
|
Departments of Physics, University of California, Berkeley, CA 94720, USA. |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Fluorescent Dyes
/
chemistry* Kinetics Light* Microscopy / methods* Models, Theoretical Proteins / analysis* |
| Grant Support | |
ID/Acronym/Agency:
|
R01-GM032543/GM/NIGMS NIH HHS; //Howard Hughes Medical Institute |
| Chemical | |
Reg. No./Substance:
|
0/Fluorescent Dyes; 0/Proteins |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: A Syrian golden hamster model recapitulating ebola hemorrhagic fever.
Next Document: Phase of atmospheric secondary organic material affects its reactivity.