Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

9-POS Board 9 APPLICATION OF SUPER RESOLUTION RADIAL FLUCTUATION (SRRF) IMAGING TO MEASUREMENT OF SINGLE-MOLECULE DNA HYBRIDIZATION KINETICS. Justin Cooper 1,2 ; 1 Andor Technology, Concord, MA, USA 2 University of Utah, Chemistry, Salt Lake City, UT, USA Super-resolution radial fluctuations (SRRF) is a novel algorithm based super-resolution imaging technique that incorporates radial intensity gradients and temporal intensity fluctuations to calculate and project SRRF images with resolution up to 2x below the diffraction limit in real time. SRRF, has been used for imaging sub-diffraction intracellular structures and for performing time-resolved super resolution measurements of live cell dynamics. However, no application to single-molecule localization and tracking techniques has been shown. Here, we characterize the capabilities of the SRRF algorithm on measuring DNA hybridization kinetics from a DNA capture surface via single-molecule localization and tracking. We find that SRRF data has some obvious advantages such as a substantial increase in the signal-to-background ratio of single-molecule data. This is a side effect of the image radially transform which yields low radially values for regions which contain randomized intensity values on the distances scales of the radiality calculation. This is particularly usefully when measuring hybridization kinetics with slow on rates which necessitate higher solution phase concentrations and resulting in higher background levels. We also find that with sparse data such as these, molecule localization can be performed by calculation of the 1 st moment centroids of the SRRF point spread functions (PSF) which can deliver spatial resolution similar to non-linear fitting of the optical PSF but with much faster computational times. Finally, we explore the ability for SRRF to resolve individual molecules in high DNA surface density conditions which result in many overlapping optical PSFs. We find that measuring single-molecule data following the SRRF radiality transform allows for resolving much higher densities of molecules than diffraction limited images thus allowing for faster collection of statistically significant population data.

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