Calibration-free counting of low molecular copy numbers in single DNA-PAINT localization clusters

Johannes Stein; Florian Stehr; Ralf Jungmann; Petra Schwille

doi:10.1016/j.bpr.2021.100032

Calibration-free counting of low molecular copy numbers in single DNA-PAINT localization clusters

Biophys Rep (N Y). 2021 Nov 10;1(2):100032. doi: 10.1016/j.bpr.2021.100032. eCollection 2021 Dec 8.

Authors

Johannes Stein¹, Florian Stehr¹, Ralf Jungmann^{1

2}, Petra Schwille¹

Affiliations

¹ Max Planck Institute of Biochemistry, Martinsried, Germany.
² Faculty of Physics, Ludwig Maximilian University, Munich, Germany.

Abstract

Single-molecule localization microscopy (SMLM) has revolutionized light microscopy by enabling optical resolution down to a few nanometer. Yet, localization precision commonly does not suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Because each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been persistently proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photophysics or on a calibration to a reference. Previously, we developed localization-based fluorescence correlation spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-PAINT (points accumulation for imaging in nanoscale topography), for the first time, to our knowledge, circumventing the necessity for reference calibrations. Here, we present an extended concept termed lbFCS+, which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting of fluorescent loci contained in individual nanoscopic volumes is achieved via precise measurement of the local hybridization rates of the fluorescently labeled oligonucleotides ("imagers") employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to 10 docking strands. For N ≤ 4 target molecules, lbFCS+ is even able to resolve integers, providing the potential to study the composition of up to tetrameric molecular complexes. Furthermore, we show that lbFCS+ allows resolving heterogeneous binding dynamics, enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from biosensing to DNA computing.