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Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM)

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 thei... Full description

Journal Title: Proceedings of the National Academy of Sciences 10/23/2012, Vol.109(43), pp.17436-17441
Main Author: Lee, S.-H.
Other Authors: Shin, J. Y. , Lee, A. , Bustamante, C.
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: ISSN: 0027-8424 ; E-ISSN: 1091-6490 ; DOI: http://dx.doi.org/10.1073/pnas.1215175109
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recordid: crossref10.1073/pnas.1215175109
title: Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM)
format: Article
creator:
  • Lee, S.-H.
  • Shin, J. Y.
  • Lee, A.
  • Bustamante, C.
subjects:
  • Sciences (General)
ispartof: Proceedings of the National Academy of Sciences, 10/23/2012, Vol.109(43), pp.17436-17441
description: 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.
language: eng
source:
identifier: ISSN: 0027-8424 ; E-ISSN: 1091-6490 ; DOI: http://dx.doi.org/10.1073/pnas.1215175109
fulltext: fulltext
issn:
  • 00278424
  • 0027-8424
  • 10916490
  • 1091-6490
url: Link


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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.

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