Super-Resolution Microscopy as a Tool for Counting Proteins in a Sub-Cellular Environment

Abstract

Super-resolution microscopy, and in particular single molecule localization microscopy (such as stochastic optical reconstruction microscopy, STORM), offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. The development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution microscopy. The challenges of molecular counting using super-resolution [3,4] can be addressed using calibration standards such as DNA origami, since they provide direct characterization for quantitative imaging when immunofluorescence is used. Here we propose to use quantitative approaches based on DNA origami calibration to study the distribution of adhesion proteins and membrane proteins. First, we focus on quantitative studies of the stoichiometry of membrane proteins (i.e membrane channels and their accessory subunits). Furthermore, we apply this method for average protein copy number estimation of adhesion proteins (i.e. vinculin) in mammalian cells and neurons. 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62. 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21. 3. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016) 4. Cella Zanacchi F. et al., DNA Origami: Versatile super-resolution calibration standard for quantifying protein copy-number, Nature Methods 14, 789–792 (2017) (2017)