Event-based Single Molecule Localization Microscopy ( eventSMLM ) for High Spatio-Temporal Super-resolution Imaging

Photon emission by single molecules is a random event with a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing a temporal change in photons and emission characteristics within a single blinking period (typically, ∼ 30 ms ) of a single molecule. This information can be used to better localize single molecules within a user-defined collection time (shorter than average blinking time) of the event detector. The events collected over every short interval of time / collection time (∼ 3 ms ) give rise to several independent temporal photon distributions ( tPSFs ) of a single molecule. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period / collection time than the entire blinking period gives rise to several realizations of the temporal PSFs ( tPSFs ) of a single molecule. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule tPSF give several position estimates of the single-molecules, leading to multiple tPSF centroids. Fitting these centroid points by a circle provides an approximate position (circle center) and geometric localization precision (determined by the FWHM of the Gaussian) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the data (photon detection events on the detector pixels) and the recorded tPSF , the estimated value is purely experimental rather than theoretical (Thomson’s formula). Moreover, the temporal nature of the event camera and tPSF substantially reduces noise and background in a low-noise environment. The method is tested on three different test samples (1) Scattered Cy3 dye molecules on a coverslip, (2) Mitochondrial network in a cell, and (3) Dendra2HA transfected live NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events (temporal change in the number of photons). Experimental results on transfected NIH3T3 cells show a localization precision of ∼ 10 nm , which is ∼ 6 fold better than standard SMLM. Moreover, imaging HA clustering in a cellular environment reveals a spatio-temporal PArticle Resolution (PAR) (2.3 l p × τ ) of 14.11 par where 1 par = 10 −11 meter . second . However, brighter probes (such as Cy3) are capable of ∼ 3.16 par . Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. The single-molecule imaging on live cells reveals temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy that is capable of high spatio-temporal particle resolution in the sub-10 par regime.