Single-molecule imaging over a large area is beneficial for understanding interlinked intracellular biophysical processes and cell-cell interaction. To study, the interrogation system requires real-time adaptability of the field-of-view (FOV). We developed a widefield nonscanning system (aSMLM) that consists of an autotunable illumination system. The 4f-autotunable optical sub-system (combination of autotunable lens and objective lens) is introduced in the illumination path to enable change of focus at the specimen plane (located at the working distance of the objective lens). The combined incident parallel beam (of wavelengths, 405 and 561 nm) is converged/diverged by the 4f subsystem, resulting in a change of focal spot at the working distance. The spot is essentially a defocussed field with an order increase in FOV (14:79 to 316:31 lm 2 ) and demonstrates better field homogeneity. However, the most important point is the tunability of the FOV in this range. A dedicated control unit is engaged to facilitate a rapid shift of focus (at a rate of 200 Hz), resulting in desirable spot-size (FOV). However, the detection subsystem is a 4f-system that collects light emerging from the specimen plane (located at the working distance of objective lens) and produces an image at the focus of tube-lens. The technique is further explored to study single-molecule (Dendra2-HA) clusters in transfected NIH3T3 cells that demonstrate its efficiency over a variable FOV. A near-uniform illumination of desired FOV is achieved along with a threefold increase in the number of detected single molecules. We anticipate that the proposed aSMLM technique may find immediate application in the emerging field of single-molecule biophysics and fluorescence microscopy.

NS2B/NS3 complex is a key protein complex essential for proteolytic activity and processing of viral polyprotein during Dengue (Denv-2) infection. The underlying mechanism involved in the early onset (first 24 hrs) of Dengue pathogenesis was studied using single molecule-based super-resolution studies to understand the Denv-2 infection. The study was conducted on transfected NIH3T3 cells using two distinct photoactivable fusion plasmid DNAs (mEos3.2-NS2B/NS3 and paGFP - NS2B/NS3). Studies demonstrated that the formation of NS2B/NS3 clusters (mEos3.2 - NS2B/NS3 and paGFP - NS2B3) on the mitochondrial network induces mitochondrial fragmentation. The NS2B/NS3 complex acts as a protease that clips specific sites of mitofusin (MFN1/2) proteins, responsible for fusion which holds the network together, disrupting the mitochondrial network. Statistical analysis of super-resolution data (images) estimates an average NS2B/NS3 cluster size of 250 ~nm with a density of 5.82 * 10^2 mol/ μm^2, and has an average of 164 molecules per cluster. Based on the present study, we hypothesize that the formation of clusters and the associated cluster related parameters are critical in promoting mitochondrial fragmentation. Overall, the single molecule-based super-resolution study helped reveal the basic mechanism of single-molecule (NS2B/NS3) clustering during the onset of Dengue viral infection. Understanding the underlying biophysical mechanism of NS2B/NS3 clustering at the single molecule level may help decipher potential drug targets and the mechanisms of action to disrupt the NS2B/NS3 clusters, which may ultimately usher the way to contain/treat Dengue viral infection.

Molecules capable of emitting a large number of photons (also known as fortunate molecules) are crucial for achieving a resolution close to single molecule limit (the actual size of a single molecule). We propose a long-exposure single molecule localization microscopy (leSMLM) technique that enables detection of fortunate molecules, which is based on the fact that detecting a relatively small subset of molecules with large photon emission increases its localization precision [Formula: see text]. Fortunate molecules have the ability to emit a large burst of photons over a prolonged time ([Formula: see text] average blinking lifetime). So, a long exposure time allows the time window necessary to detect these elite molecules. The technique involves the detection of fortunate molecules to generate enough statistics for a quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 and 11 nm toward single-molecule-limit (far from diffraction-limit) for an exposure time window of 60 and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33% and 3.43% molecules are found to emit in 30–60  ms and >60 ms, respectively, when compared to single molecule localization microscopy (SMLM). The long exposure has enabled better visualization of the Dendra2HA molecular cluster, resolving sub-clusters within a large cluster. Thus, the proposed technique leSMLM facilitates a better study of cluster formation in fixed samples. Overall, leSMLM technique offers a spatial resolution improvement of ~ 10 nm compared to traditional SMLM at the cost of marginally poor temporal resolution.

Molecules capable of emitting a large number of photons (also known as fortunate molecules) are crucial for achieving resolution close to a single molecule limit (the actual size of a single molecule). We propose a long-exposure single molecule localization microscopy (leSMLM) technique that enables detection of fortunate molecules, which is based on the fact that detecting a relatively small subset of molecules with large photon emission increases its localization (~ r0/√N). Fortunate molecules have the ability to emit a large burst of photons over a prolonged time (> average triplet-state lifetime). So, a long exposure time allows the time window necessary to detect these elite molecules. The technique involves the detection of fortunate molecules to generate enough statistics for a quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 nm and 11 nm towards Single-molecule-limit (away from diffraction-limit) for an exposure time window of 60 ms and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33% and 3.43% molecules are found to emit in 30-60 ms and 60-90 ms, respectively, when compared to SMLM. The long exposure has enabled better visualization of Dendra2HA molecular cluster, with sub-clusters within a large cluster. Thus, the proposed technique (leSMLM) facilitates a better study of cluster formation in fixed samples. Overall, the method enables better spatial resolution at the cost of relatively poor temporal resolution.

Single-molecule localization microscopy can decipher fine details that are otherwise not possible using diffraction-limited microscopy. Often the reconstructed super-resolved image contains unwanted noise, random background and is prone to false detections. This cause spurious data that necessitates several trials, multiple experimentations, and repeated preparation of specimens. Moreover, this is not suitable for experiments that require time-lapse imaging and real-time microscopy. To overcome these limitations, we propose a technique (corrSMLM) that can recognize and detect fortunate molecules (molecules with long fluorescence cycles) from the recorded data. The technique uses the correlation between two or more consecutive frames to extract fortunate molecules that blink for longer than the standard blinking time. Accordingly, strongly-correlated spots (single molecule signatures) are compared in consecutive frames, followed by data integration (mean centroid position and the total number of photons) and estimation of critical parameters (position and localization precision). The technique addresses two major problems that plague SMLM : (1) random noise due to false detection that contributes to strong background, and (2) poor localization precision offered by standard SMLM techniques. On the brighter side,corrSMLMallows only fortunate molecules to represent the super-resolved image, thereby suppressing the background and improving localization precision by a factor of 2-4 times as compared to standard SMLM. To substantiate,corrSMLMis used for imaging fixed cell samples (Dendra2-Actin and Dendra2-Tubulin transfected NIH3T3 cells). Results show multi-fold reduction in noise and localization precision with a marked improvement in overall resolution and SBR. We anticipatecorrSMLMto improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.