Advancing optical nanoscopy by combining event-based sensors with modulation-enhanced single-molecule localization microscopy

Non-invasive, high-resolution imaging of biological systems often requires optical microscopy. Since overcoming the diffraction barrier, optical nanoscopy, or super-resolution microscopy, with up to ~1-10 nm resolution can be used for imaging immobilized or live biological samples. The final frontier of optical nanoscopy is high-throughput, true molecular imaging (~1 nm precision) in dynamic, high-mobility systems. Currently, the most promising nanoscopy methods employ modulation-enhanced localization microscopy. However, these are compromised either in throughput or in spatiotemporal precision. This trade-off is due to a sensor choice focused on rich temporal (realizing high localization precision) or rich spatial (realizing high throughput) information. A novel sensor architecture, event-based sensors, merges these choices by measuring changes in photon flux on a megapixel-sized chip with asynchronously recorded microsecond-precise timestamped events. With ES-FLUX, I will combine patterned sinusoidal illumination patterns with event-based single-molecule localization microscopy. This will break the stalemate between high throughput and high precision in modulation-enhanced localization microscopy. The spatiotemporal single-molecule event patterns will be analysed separately in the temporal and in the spatial domains, and as such ES-FLUX will improve the resolution compared to traditional single-molecule localization microscopy. It paves the way for molecular precision whilst allowing the high experimental throughput associated with full-frame camera acquisition. Further improvements in the computational analysis and optomechanical setup expands ES-FLUX towards single-particle tracking and spectral multiplexing. In conclusion, ES-FLUX merges modulation-enhanced single-molecule localization microscopy with event-based sensor acquisition and as such expands the boundaries of optical nanoscopy.