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

Sang Hyuk Lee, Jae Yen Shin, Antony Lee, Carlos Bustamante

Research output: Contribution to journalArticlepeer-review

211 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)17436-17441
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number43
DOIs
StatePublished - Oct 23 2012
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Fluorescence blinking
  • Single molecule counting
  • Super-resolution optical microscopy

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