Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging

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 a minimal change in the system hardware (requiring 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 the 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 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.

The Anti-inflammatory Drug Leflunomide Inhibits NS2B3 Cluster Formation During Dengue Viral Infection as Revealed by Single Molecule Imaging

“…A homemade scanSM LM system is built to carry out the study, the details of which can be found in Ref. [12]. First, the system is operated in an epifluorescence mode to identify a transfected cell, as shown in Fig.…”

“…To overcome this, single-molecule super-resolution microscopes are ideal since it can resolve features beyond the diffraction limit ( ∼ λ/ 2). Of all the existing super-resolution techniques (such as STED, structured illumination, and its recent variants), fluorescence photoactivation localization microscopy (FPALM) and scanning SMLM ( scanSM LM ) holds promise since it can quantify individual molecules and their collective dynamics with high temporal resolution in a cell volume [24][12]. The technique uses photoactivable fluorescent probes that is activated by a high-frequency laser and convert a small fraction of inactive fluorophore to active form.…”

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

“…6A. Prominent clusters are identified using DBSCAN algorithm [41] and the individual clusters are colored (see, Fig. 6A).…”

Aberration correction for deformable mirror based remote focusing enables high-accuracy whole-cell super-resolution imaging

“…In an approach known as remote focusing, fast 3D imaging of thick samples can be achieved by dynamically adjusting the focal plane without physically moving the objective lens or sample [19][20][21][22][23]. This process can be executed at rates of a few KHz using a DM or other variable optical elements [19,24,25], avoiding the need for physical movement of sample or objective. In SMLM, this method has demonstrated the capacity to record volumetric data of whole cells up to 10 µm while maintaining axial sample stabilization using a standard widefield microscope equipped with a DM [20].…”