Single-molecule switching (SMS) nanoscopy techniques like STORM/(F)PALM circumvent the diffraction limit to enable 3D fluorescence imaging with resolutions in the range of 10-75 nanometers, an order of magnitude better than conventional light microscopes. This increased spatial resolution affords many opportunities to investigate sub-cellular function and organization (for example of the interphase nucleus) but is achieved by sacrificing temporal resolution - typically requiring several minutes to reconstruct a single super-resolution image. While our lab recently implemented high-speed SMS by increasing the acquisition rate 16-fold, conventional analysis pipelines cannot handle this data volume. We have developed a framework for real-time single-molecule localization analysis and distributed post-localization analysis which enables high-throughput SMS. By imaging over 11,000 cells and performing automated analysis, we demonstrate that the data and analysis bottlenecks of SMS techniques are solved, allowing SMS to be used as a valid tool for large-scale quantitative hypothesis testing.

1. David Baddeley (d.baddeley@auckland.ac.nz)

1. Joerg Bewersdorf (joerg.bewersdorf@yale.edu)

N/A