title: Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM).
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.
authors.affiliation: Departments of Physics, University of California, Berkeley, CA 94720, USA.
authors.forename: Sang-Hyuk
authors.initials: SH
authors.lastname: Lee
authors.forename: Jae Yen
authors.initials: JY
authors.lastname: Shin
authors.forename: Antony
authors.initials: A
authors.lastname: Lee
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008853:Microscopy / Q000379:methods*; D008962:Models, Theoretical; D011506:Proteins / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins