한빛사 논문
Abstract
Sang-Hyuk Leea,b,1, Jae Yen Shinc,1, Antony Leea, and Carlos Bustamantea,b,c,d,e,2
aDepartments of Physics,
dMolecular and Cell Biology, and
eChemistry, University of California, Berkeley, CA 94720;
bQB3 Institute, University of California, Berkeley, CA 94720; and
cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720
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.
super-resolution optical microscopy, single molecule counting, fluorescence blinking
Footnotes
1S.-H.L and J.Y.S. contributed equally to this work
2To whom correspondence should be addressed.
Author contributions: S.-H.L., J.Y.S., and C.B. designed research; S.-H.L. and J.Y.S. performed research; S.-H.L., J.Y.S., and A.L. analyzed data; and S.-H.L., J.Y.S., A.L., and C.B. wrote the paper.
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