Basumatary, J and Baro, N and Joshi, P and Mondal, PP (2023) Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging. In: Communications Biology, 6 (1).
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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. We introduce a single molecule-based scanning SMLM (s c a n S M L M) system that enables rapid volume imaging. Along with epi-illumination, the system employs a scanning-based 4f detection for volume imaging. The 4f system comprises a combination of an electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of an electrically-tunable lens (ETL) in a 4f detection system, the selectivity of the axial object plane is achieved, for which the image forms in the image/detector plane. So, in principle, one can scan the object volume by just altering the aperture of ETL. Two schemes were adopted to carry out volume imaging: cyclic scan and conventional scan. The cyclic scheme scans the volume in each scan cycle, whereas plane-wise scanning is performed in the conventional scheme. Hence, the cyclic scan ensures uniform dwell time on each frame during data collection, thereby evenly distributing photobleaching throughout the cell volume. With a minimal change in the system hardware (requiring the addition of an ETL lens and related electronics for step-voltage generation) in the existing SMLM system, volume scanning (along the z-axis) can be achieved. To calibrate and derive critical system parameters, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, s c a n S M L M is employed to visualize the architecture of actin-filaments and the distribution of Meos-Tom20 molecules on the mitochondrial membrane. The technique is further exploited to understand the clustering of Hemagglutinin (HA) protein single molecules in a transfected cell for studying Influenza-A disease progression. The system, for the first time, enabled 3D visualization of HA distribution that revealed HA cluster formation spanning the entire cell volume, post 24 hrs of transfection. Critical biophysical parameters related to HA clusters (density, the number of HA molecules per cluster, axial span, fraction of clustered molecules, and others) are also determined, giving an unprecedented insight into Influenza-A disease progression at the single-molecule level. © 2023, Springer Nature Limited.
| Item Type: | Journal Article |
|---|---|
| Publication: | Communications Biology |
| Publisher: | Nature Research |
| Additional Information: | The copyright for this article belongs to the Authors. |
| Department/Centre: | Division of Physical & Mathematical Sciences > Centre for Cryogenic Technology Division of Physical & Mathematical Sciences > Instrumentation Appiled Physics |
| Date Deposited: | 12 Dec 2023 03:44 |
| Last Modified: | 12 Dec 2023 03:44 |
| URI: | https://eprints.iisc.ac.in/id/eprint/83344 |
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