Optimising fluorophore performance in single molecule localisation microscopy using novel SPAD imagers

Single molecule localisation microscopy (SMLM) requires sequential switching on and off a small subset of fluorophores to enable the localisation of individual fluorophores and subsequent generation of a super-resolution image. The choice of fluorescent probe and buffer conditions can impact on the quality of super-resolution images. Additionally, attainment of good quality images is hindered by the uncertainty in the rate at which images should to be acquired, in order to capture the millisecond fluorescence event for each fluorophore, with suitable signal-to-noise ratio, to enable their position to be determined with the highest accuracy and precision.
CMOS SPAD (single photon avalanche diode) image sensors allow for fast frame rates (>10k frames/s) and single photon resolution, which potentially allow for higher accuracy and precision of fluorphore position than traditional EMCCD or sCMOS imagers. These novel, fast CMOS SPAD imagers are used to characterise the switching properties of a selection of the most commonly used fluorophores in SMLM applications in a range of buffer conditions. This information allows for more accurate, quantitative characterization of the switching properties of individual fluorophores and can facilitate optimisation of imaging parameters for acquiring SMLM images using the traditional EMCCD.