Biology subjects

Pant, N.

Publications and source records attributed to Pant, N..

4 recordsLinked to original sources

bioRxiv2025

Chiral Single Molecule Localization Microscopy (chiralSMLM)

Chiral fingerprints are unique to a molecule and convey information related to binding sites, conformation, and its immediate chemical environment. Deciphering this information is expected to aid a better understanding of the biological processes (protein trafficking, kinetics, and aggregation) in a cellular environment at the scale of a single molecule. Here, we present a new optical technique called chiralSM LM, to selectively detect chiral enantiomers (both left-handed and right-handed) of single molecules and generate a super-resolved map to understand the role of chirality associated with the biological processes in a cell. Accordingly, calibration, characterization, and optimization of the system are carried out using a polarized light source (linear polarized, left and right circularly polarized) and by recording known structures (Actin filaments) in a cell. The system is employed to carry out cell transfection studies on two different disease models (Hemagglutinin protein for Influenza type-A and NS3 protein for Dengue type-2) to understand the role of molecular chirality on disease specific biological processes leading to clustering. Single-molecule cluster analysis revealed that left-handed Dendra2-HA and Dendra2-NS3 molecules have a larger footprint, suggesting the role of chiral molecules in promoting cluster formation. In addition, the presence of left-handed molecules at the cluster-periphery is perplexing. This is interesting, as it demonstrates the active role of single molecule handedness (left or right) during protein clustering in a transfected cell. The new classification of single molecules purely based on their chiral nature is expected to advance single-molecule imaging and provide new insights in disease biology. Summary & SignificanceThe chirality of single protein molecules is crucial to its functioning. This provides a new perspective on understanding biological functions, based solely on the chiral nature of proteins, and deciphers its role in a cellular processes. We developed a chiralSM LM microscopy system to access the role of chirality on the clustering behavior of viral proteins (HA for Influenza type-A and NS3 in Dengue type-2) in a cell. Hence, the technique offers a new approach to quantify chirality aided molecular interactions in disease biology.

biophysics↗

bioRxiv2024

TRapping and IMaging (TRIMing) of Cells/Multicellular Organisms in Free Living Environment Enabled by Adaptive Lightsheet Optical Tweezer (aLOT)

To be able to trap and image in a live cell / organism on the go is an incredible feat and paves the way for immobilization-free interrogation. This is a step towards the interrogation of cells / live species in their natural environment. To facilitate, a TRIMing technique primarily based on an adaptive lightsheet optical tweezer (aLOT) system is proposed. The TRIMing technique combines the benefits of touch-free optical tweezing and high-resolution imaging. The entire system is built on a single platform for rapid interrogation of freely moving live biological specimens. The trapping system combines an electrical-tunable lens (ETL), cylindrical lens, and an objective lens to generate adaptive PSF. The ETL (in the beam-expander) adaptively changes the beam cross-section (to either a parallel beam or converging point-beam) entering the back-aperture of cylindrical lens, resulting in a point or a line spot at the focus. An objective lens placed at the focus of a cylindrical lens converts the spot to a tightly focused diffraction-limited lightsheet or point PSF. Depending on the object type (spherical or elongated), the system can flip between point and sheet PSF at a rate of 200 Hz. The system is integrated to a separate fluorescence arm to enable the imaging of trapped objects (cells or organisms). The TRIMing system operates in a brightfield mode to optically trap using point / sheet PSF and subsequently switched to fluorescence mode for imaging. The potential of the system is demonstrated by trapping live specimens (HeLa cells and C. elegans labelled with Bodipy dye) and imaging them in a freely moving environment. Characterization shows a point and sheet PSF size of, 43.42 m2 and 70.5 x 4.9m2 with a trap stiffness of 1.15 x 10-3 pN/nm and 0.46 x 10-3 pN/nm, respectively. Fluorescently-labelled live specimens were investigated that showed the random distribution of organelles (lipid droplets) both in cells and C. elegans. The TRIMing system demonstrated a resolution of < 0.7m, a contrast of {approx} 0.84, a SNR of {approx} 11 dB. This allows a good combination of rapid trapping and high-quality imaging. In addition, the system allows near real-time determination of critical biophysical parameters, such as organelle size of 1.01 m (in cells) and 1.29 m (in C. elegans) with a density of 0.021#/m2 and 0.039#/m2, respectively. The number of lipid droplets are found to be nearly double for C. elegans as compared to HeLa cells. These parameters are directly linked to the physiological state of live biological species. Overall, the developed TRIMing system allows high-quality imaging of live specimens in a free living environment. Statement of SignificanceThe ability to image live specimens in a free-living environment is phenomenal. The existing techniques often constrain/fix/anesthetize these organisms to image their physiological state. This comes with a lot of conditioning and directly affects the physiological state or developmental process in biological species, especially the brain undergoing neuronal activity. The proposed TRIMing technique elevates this requirement by optically trapping the moving object and simultaneously imaging the internal organelles with high resolution in a free environment. The technique is expected to have widespread applications in diverse disciplines ranging from fundamental cell biology to optical physics.

biophysics↗

bioRxiv2023

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

Photon emission by single molecules is a random event with a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing a temporal change in photons and emission characteristics within a single blinking period (typically, [~] 30 ms) of a single molecule. This information can be used to better localize single molecules within a user-defined collection time (shorter than average blinking time) of the event detector. The events collected over every short interval of time / collection time ([~] 3 ms) give rise to several independent temporal photon distributions (tPSFs) of a single molecule. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period / collection time than the entire blinking period gives rise to several realizations of the temporal PSFs (tPSFs) of a single molecule. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule tPSF give several position estimates of the single-molecules, leading to multiple tPSF centroids. Fitting these centroid points by a circle provides an approximate position (circle center) and geometric localization precision (determined by the FWHM of the Gaussian) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the data (photon detection events on the detector pixels) and the recorded tPSF, the estimated value is purely experimental rather than theoretical (Thomsons formula). Moreover, the temporal nature of the event camera and tPSF substantially reduces noise and background in a low-noise environment. The method is tested on three different test samples (1) Scattered Cy3 dye molecules on a coverslip, (2) Mitochondrial network in a cell, and (3) Dendra2HA transfected live NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events (temporal change in the number of photons). Experimental results on transfected NIH3T3 cells show a localization precision of [~] 10 nm, which is [~] 6 fold better than standard SMLM. Moreover, imaging HA clustering in a cellular environment reveals a spatio-temporal PArticle Resolution (PAR) (2.3lp x{tau} ) of 14.11 par where 1 par = 10-11 meter.second. However, brighter probes (such as Cy3) are capable of [~] 3.16 par. Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. The single-molecule imaging on live cells reveals temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy that is capable of high spatio-temporal particle resolution in the sub-10 par regime.

biophysics↗

bioRxiv2023

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

A prerequisite for Dengue viral infection is the clustering of NS2B3 viral protein in the infected cell. This calls for drugs capable of reversing the biological processes leading to the declustering of NS2B3 viral complex. In this work, we report a new drug (leflunomide) that shows reversal of NS2B3 clustering, post 24 hours of cell transfection with a recombinant probe (Dendra2-NS2B3) containing the viral complex of interest (NS2B3). To study, we constructed a photoactivable recombinant plasmid for visualizing the activity of the target protein-of-interest (Dendra2-NS2B3). This enabled a better understanding of the underlying biological processes involved in Dengue and the role of NS2B3. The study was performed in a cellular system by transfecting the cell (NIH3T3 -mouse fibroblast cell line), followed by drug treatment studies. A range of physiologically relevant concentrations (250 nM - 10 M) of the FDA-approved drug (leflunomide) was used. The single molecule super-resolution microscopy (scanSM LM) study showed declustering of NS2B3 clusters for concentrations > 250 nM and near complete disappearance of clusters at concentrations > 5 M . Moreover, the associated critical biophysical parameters suggest a substantial decrease in clustered molecules (from 53.2 {+/-} 1.77% for control to 14.89 {+/-} 4.80% at 250 nM, and further reduction to 10.55 {+/-} 2.91% at 500 nM). Moreover, the number of clusters reduced from 46 {+/-} 15 to 13 {+/-} 4, and the number of molecules per cluster decreased from 133 {+/-} 29 to 62 {+/-} 3, with a depletion in large clusters (from 24 to 12). The parameters collectively indicate the clustering nature of NS2B3 viral protein during the infection process at a cellular level and the effect of leflunomide in declustering. The results supported by statistical analysis suggest strong declustering promoted by leflunomide, which holds the promise to contain/treat dengue viral infection. Statement of SignificanceThe fact that there is no approved antiviral approach for Dengue makes it life-threatening and calls for ways to tackle viral infection. Hence, understanding Dengue biology at a single molecule level plays a vital role. In the present super-resolution study, we noted the formation of key viral protein (NS2B3) clusters post 24 hours of transfection in a cellular system. We identified a repurposed FDA-approved drug (Leflunomide) that inhibits the clustering process and promotes declustering at higher drug concentrations. This may become the basis of future studies, which may have therapeutic potential against Dengue.

biophysics↗