@article {Basumatary2022.04.01.486682, author = {Jigmi Basumatary and Neptune Baro and Prakash Joshi and Partha Pratim Mondal}, title = {Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging}, elocation-id = {2022.04.01.486682}, year = {2022}, doi = {10.1101/2022.04.01.486682}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Over the last decade, single molecule localization microscopy (SMLM) has developed into a set of powerful techniques that has improved spatial resolution over diffraction-limited microscopy and has demonstrated the ability to resolve biological features at molecular scale. We introduce a single molecule based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Using a standard widefield illumination, the system employs a scanning based detection 4f-sub-system suited for volume interrogation. The 4f system comprises of a combination of electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of electrically-tunable lens (ETL) in a 4f detection system, the selectivity of axial (Z) plane can be achieved in the object plane, for which the corresponding image forms in the image/detector plane. So, in-principle one can scan the object volume by just changing the aperture of ETL. To carry out volume imaging, a cyclic scanning scheme is developed and compared with conventional scanning routinely used in SMLM. The scanning scheme serves the purpose of distributing photobleaching evenly by ensuring uniform dwell time on each frame for collecting data (single molecule events) throughout the specimen volume. With minimal change in the system hardware (requiring an addition of ETL lens and related hardware for step-voltage generation and time synchronization) in the existing SMLM system, volume scanning can be achieved. To demonstrate, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to understand clustering of HA single molecules in a transfected cell (Influenza A disease model). The system for the first time enables visualization of HA distribution in a 3D cells that reveal its clustering across the cell volume. Critical biophysical parameters related to HA clusters (density, $\#$HA/cluster and cluster fraction) are also determined for a single NIH3T3 cell transfected with photoactivable Dendra2-HA plasmid DNA.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2022/04/03/2022.04.01.486682}, eprint = {https://www.biorxiv.org/content/early/2022/04/03/2022.04.01.486682.full.pdf}, journal = {bioRxiv} }