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

Abstract

Photon emission by single molecules is a random event that has a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing the arrival time of photons and their distribution (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 (~ 3 ms) give rise to several independent photon distributions (PSFs) of single molecules in the event camera. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period than the entire blinking period gives rise to several realizations of single-molecule PSFs. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule PSF give several position estimates of the single-molecules, leading to multiple PSF centroids. Fitting these centroid points by a circle gives an approximate position (circle center) and geometric localization precision (circle area) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the photon detection events and the recorded PSF, the estimated value is purely experimental rather than theoretical (Thomson's formula). Moreover, this eliminates the need for noise calculation and background estimation. 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 NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events / photons. Experimental results on transfected NIH3T3 cells show a localization precision of ~ 10 nm, which is ~ 3.5 fold better than standard SMLM. Results reveal a spatio-temporal resolution (lp × t) of 122.5p (where, p=10-12meter.second) (measured in terms of localization precision). Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. Moreover, single-molecule imaging on live cells reveals the temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over a period of 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy capable of high spatio-temporal super-resolution microscopy (in the range ~ 1p = 10-12meter.sec).