Single Molecule Based Spatio-temporal Super-resolution Microscopy for Visualizing Single Molecule Dynamics in Cellular System

Abstract

Over the last decade, single-molecule localization microscopy (SMLM) has developed into a set of powerful techniques that have improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features down to a few tens of nanometers. This thesis describes new approach of imaging and detecting single molecule to address important biological questions. Chapter \ref{chp1} presents a brief introduction to diffraction of light, resolution limit in optical microscopy, photo luminescence process and historic development of optical microscopy. Moreover, chapter discuss SMLM technique primarily focused on Fluorescence/Photo Activation Localization Microscopy (f/PALM) technique. Chapter \ref{chp2} presents fast imaging of single molecules using an sCMOS detector in a traditional epifluorescence SMLM called temporally-resolved SMLM. Realizing that a substantial fraction of single molecules emit photons for time scales much shorter than the average blinking period, proposed approach accelerates data collection to capture these fast emitters. Thorough analysis shows $\approx$2.76-fold improvement in the temporal resolution that comes with a sacrifice in spatial resolution, and a particle resolution shift PAR-shift (in terms of localization precision) of $\approx 11.82 nm$ compared to standard SMLM. The benefits of the reported approach are demonstrated by imaging HA cluster in a live NIH3T3 cell. Chapter \ref{chp3} presents newly developed scanSMLM system, a 3D Single Molecule Imaging approach, which is implemented on an epifluorescence SMLM. 3D imaging of the specimen is achieved by tuning the focal length of the electrically tunable lens (ETL) installed in the $4f$ detection sub-system. The developed system is thoroughly calibrated and standardized using nano-bead and demonstrated by imaging organelles (mitochondria and actin filaments) in a fixed NIH3T3 cell. Furthermore, the developed system is used to image the distribution of influenza viral protein HA (hemagglutinin) in fixed NIH 3T3 cells, 24hrs post-transfection. Chapter \ref{chp4} presents new realtime single-molecule volume imaging using scanSMLM. Realtime analysis in scanSMLM is performed using a fast sCMOS detector in a single-core CPU. sCMOS detector functionality is accessed using SDK3 written in Matlab. Cyclic volume scanning of the specimen via ETL tuning is synchronized with real time data acquisition. Analysis demonstrate realtime data acquisition rate of $10Hz$. The technique was demonstrated by imaging HA cluster in live cell. Chapter \ref{chp5} presents event-based detection of single molecule. In traditional SMLM, the molecule PSF is recorded by a fixed frame rate detector. The fixed frame of currently used detector technology limits the information of sporadic fluorescence emission of scholastically activated single molecules. Unlike frame-based detectors, in event-based detectors, each pixel is independent and responds to the change in intensity. The temporal resolution in event-based cameras is significantly higher than the existing frame rate based detectors and is limited by circuit latency only. The output of the event detector is a stream consisting of positive and negative events. This stream of event can be collected for fixed time window(shorter than the average blinking period of single molecule) to construct and locate event PSF. Initially, response of an event-based detector to an ON/OFF event of a continuous laser is demonstrated. Subsequently, event-based detection of Cy3 molecules and event-based localization scheme is proposed. Further, even detector is employed to image mitochondria for mEosTom20 transfected NIH3T3 cell. Moreover, investigation of viral protein (Dendra2HA) is carried out both in fixed and live NIH3T3 cells 24hrs post-transfection. Chapter \ref{chp6} concludes the thesis summary and discusses the future direction of the findings.