Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM).
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TLDR
A kinetic model is introduced to describe blinking and it is shown that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra 2 a better photoactivated localization microscopy tag than m Eos2 for molecular counting.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.read more
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References
More filters
Journal ArticleDOI
Imaging intracellular fluorescent proteins at nanometer resolution.
Eric Betzig,George H. Patterson,Rachid Sougrat,O. Wolf Lindwasser,Scott G. Olenych,Juan S. Bonifacino,Michael W. Davidson,Jennifer Lippincott-Schwartz,Harald F. Hess +8 more
TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Journal ArticleDOI
Precise nanometer localization analysis for individual fluorescent probes
TL;DR: A localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations), and results show good agreement with the derived precision equation.
Journal ArticleDOI
Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells.
Yuichi Taniguchi,Paul J. Choi,Gene-Wei Li,Huiyi Chen,Mohan Babu,Jeremy Hearn,Andrew Emili,X. Sunney Xie +7 more
TL;DR: System-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size.
Journal ArticleDOI
Subunit counting in membrane-bound proteins.
TL;DR: A single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest is described.
Journal ArticleDOI
Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
Fedor V. Subach,George H. Patterson,Suliana Manley,Jennifer M. Gillette,Jennifer Lippincott-Schwartz,Vladislav V. Verkhusha +5 more
TL;DR: Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two- color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM).