Biology subjects

Mondal, P. P.

Publications and source records attributed to Mondal, P. P..

16 recordsLinked to original sources

bioRxiv2025

Super-resolution Fluorescence Microscopy Reveals Densely Packed EboV Membrane Glycoprotein Clusters in the Cell Plasma-membrane During Ebola Infection

The glycoprotein (GP) of Ebola virus plays a central role in the viral entry into host cells. The detailed study of Ebola glycoprotein in the cellular system is unknown, mainly due to the resolution limit ([~]{lambda} /2) of optical fluorescence microscopes. To overcome this, we propose to use single-molecule localization microscopy (SMLM), which enables the study of the role of glycoproteins at both the single-molecule and ensemble levels. Accordingly, a photoactivable recombinant probe Dendra2 - EboV Glyc is developed by linking Ebola glycoprotein with Dendra2. The transient expression of photoactivable recombinant protein in NIH3T3 cells indicated the dominant presence of glycoprotein in the plasma membrane with regions of densely packed glycoproteins. Super-resolution single-molecule imaging gives direct proof of glycoprotein clusters in the plasma membrane. The corresponding single-molecule analysis using DBSCAN clustering algorithm reveals a dense packing of glycoprotein with an estimated average density of 5345 mol/{micro}m2 compared to the density in other regions of the membrane. The number of glycoproteins is estimated to be as high as 642 molecules per cluster and clusters spread area of {approx} 0.1219 {micro}m2 after 48 hrs post-transfection. Noting that glycoprotein is involved in cellular attachment, endosomal entry, and membrane fusion, the formation of glycoprotein clusters in the membrane is significant in the progression of Ebola pathogenesis. A drug capable of dispersing the glycoproteins clusters could be of therapeutic potential to lessen the intensity of infection and to slow down the disease progression. Statement of SignificanceThe fact that powerful fluorescence microscopes (such as SMLM) are available is a boon to disease biology, specifically to study the function of viral proteins at the cellular level. Ebola, one of the nocturnal viral diseases, is a threat and calls for study at the single-molecule level to understand the dynamics (kinetics and ensemble) of glycoprotein during viral infection. In this respect, the super-resolution microscopy techniques may help reveal the biological mechanism behind the infection process at the single-molecule level. A photoactivable fluorescent probe (Dendra2 - EboV Glyc) containing the gene-of-interest (EboVGP) is developed, and transfection studies are carried out, showing densely packed glycoprotein clusters in the cell plasma membrane with specific density, number, and size. We believe that dispersing these glycoprotein protein clusters could be of therapeutic potential.

biophysics↗

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↗

bioRxiv2024

Planar Optical Tweezer Trap (2D-LOT) System Realized by Light Sheet Illumination & Orthogonal Widefield Detection

We report the realization of the first planar optical tweezer trap system by a sheet of light. To visualize the trapping of the target object (dielectric bead or live cell) in a plane, an orthogonal widefield detection is employed. The planar / two-dimensional lightsheet optical tweezer (2D-LOT) sub-system is realized in an inverted microscopy mode with illumination from the bottom. A 1064 nm laser (power [~] 500mW) is expanded and directed to a combination of cylindrical lens and high NA objective lens to generate a tightly-focused diffraction-limited light sheet. The object to be trapped is injected in the specimen chamber (consists of two coverslips placed at a distance of {approx} 1 mm) using a syringe. The solution containing the objects stayed in the chamber due to the surface tension of the fluid. The illumination of trap-laser light is along Z-direction (with coverslip along XZ-plane) whereas, the detection is achieved perpendicular to the coverslip (along Y-axis). The orthogonal detection is employed to directly visualize the trapping in a plane. To better visualize the specimen, a separate white light illumination sub-system is used. The characterization of system PSF estimates the size of light sheet trap PSF to be, 2073.84 {micro}m2 which defines the active trap region / area. Beads are tracked on their way to the trap region for determining the trap stiffness along Z and X i.e, kz = 1.13 {+/-} 0.034 pN/{micro}m and kx = 0.74 {+/-} 0.021 pN/{micro}m. Results (image and video) show real-time trapping of dielectric beads in the trap zone (2D plane) generated by the light sheet. The beads can be seen getting trapped from all directions in the XZ-plane. Prolonged exposure to the light sheet builds up a 2D array of beads in the trap zone. Similar experiments on live NIH3T3 cells show cells trapped in the 2D trap. The potential of the planar trap lies in its ability to confine objects in two dimensions, thereby opening new kinds of experiments in biophysics, atomic physics, and optical physics. Statement of SignificanceThe ability to trap and confine objects in two dimensions / a plane is an incredible feat that paves the way for new experiments in physical and biological sciences. Currently, no technique can achieve two-dimensional trapping of objects. Such a trap is realized by a sheet of light generating a near-rectangular potential trap-zone, enabling tweezing confined to a plane. This is unlike existing point-focus-based tweezers that are capable of point-potential well. The technique is expected to have widespread applications in science and engineering.

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↗

bioRxiv2023

Orientation Single Molecule Localization Microscopy (oSMLM) for Decoding Orientation of Single Molecule

Standard SMLM facilitates the reconstruction of super-resolution map (both location and localization precision) of the target single molecules. In fact, single molecule data does provide information related to the orientation of single molecules, which can be derived from the knowledge of PSF shape and its direction. This information is vital to probe the sub-domain of macromolecules that undergo orientation and conformational changes and provides essential clues on their catalytic activity. Accessing this information in real-time opens up a powerful new window to look into the link between the orientation of macromolecules and the output function. Here, we decode the orientation of single molecules from the knowledge of PSF shape and its direction. The method is primarily based on field-dipole interaction and the fact that the distribution of emitted photons strongly depends on the orientation of the dipole (fluorophore) with respect to the polarization of light. Accordingly, the photon emission from the specimen and the resultant PSF distribution model is developed. Computational studies show changes in the shape and orientation of the recorded PSF (in the image / detector plane). Specifically, a set of three distinct distributions (Gaussian, bivariate-Gaussian and skewed-Gaussian) are recognized from the study, apart from a superset of all possible (a total of 16) distributions. Experiments were conducted on Dendra2-Actin and Dendra2-HA transfected cells that validate the emission model. We report a localization precision of[~] 20 nm and an orientation precision of {+/-}5{degrees}. In addition, the distinct orientation of single molecules is noted for Actin and HA in a cell (Influenza type-A model). Further analysis suggests a preferred directional distribution of Dendra2-Actin single molecules, while Dendra2-HA molecules seem to be randomly oriented in a cluster. The availability of orientation information in SMLM without the need for additional optics adds a new feature, which can be explored to reveal the state of a single molecule (orientation and conformational changes) in cellular sub-domains / partitions. The study implies that the orientation of single molecules that has more profound implications for the functioning of macromolecules. The orientation information revealed by oSM LM technique gives it a wide-spread appeal and expands the reach of localization microscopy.

biophysics↗

bioRxiv2022

Correlation Single Molecule Localization Microscopy (corrSMLM) Detects Fortunate Molecules for Better Localization Precision and High Signal-to-Noise Ratio

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 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, corrSMLM allows only fortunate molecules contribute to 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, corrSMLM is 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 anticipate corrSMLM to improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.

biophysics↗

bioRxiv2022

Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

Understanding viral proteins at single-molecule level and visualizing their dynamics in a cellular environment is challenging. This calls for sophisticated microscopy techniques (such as SMLM) and image-based study that can reveal details with the precision of a single-molecule. Specifically, NS2B3 is recognized as a critical protein complex responsible for proteolytic activity and processing of viral polyprotein during dengue type 2 (Denv - 2) infection. Using single-molecule-based super-resolution imaging, we study the dynamics of NS2B3 protein, its kinetics and its interaction with cell organelles. Two distinct photoactivable recombinant plasmids (mEos-NS2B3 and PAGFP - NS2B3) and a fluorescent recombinant plasmid (eGFP-NS2B3) were constructed to exemplify the role of NS2B3 protein complex. The study was conducted on NIH3T3 cells and optimized transfection protocol was developed. Studies confirmed the formation of NS2B3 clusters on the mitochondrial network. Statistical analysis of super-resolution data (images) helped determine cluster dynamics and facilitated the estimation of critical biophysical parameters (such as, cluster density, number of molecules / cluster and its spread). Results indicate an average NS2B3 cluster area / spread of [~] 0.050 {micro}m2 with a density of 3500mol./ {micro}m2, and an average of [~] 120 NS2B3 molecules per cluster. In addition, regional analysis suggests a direct positive correlation between NS2B3 cluster formation (single-molecule localization microscopy study) and fragmentation of mitochondrial network (confocal microscopy study). To further exemplify, we have carried out time-lapse imaging to visualize formation of NS2B3 clusters and its dynamics. The corresponding cluster parameters (#clusters, #mol/cluster and cluster area) suggests an increase in average #mol/cluster and cluster area. For the first time, single-molecule-based super-resolution imaging study helped reveal the dynamics of NS2B3 clusters in a cellular system. Understanding the underlying dynamics of NS2B3 clustering at the single-molecule level may help to decipher potential drug targets and the ways to disrupt the NS2B3 clusters. The image-based study has immediate implications in the broad field of single molecule imaging, fluorescence microscopy and disease biology. Statement of SignificanceThe arrival of single-molecule super-resolution imaging techniques and image-based study have advanced the field of cell biology, and our understanding of sub-cellular processes with single-molecule precision. Here, we report the first ever application of super-resolution imaging to visualize NS2B3 clusters in a cellular system that directly links to the fragmentation of mitochondrial network. To facilitate the study, two new photoactivable probes (mEos - NS2B3 and P AGF P- NS2B3) with key protein complex, NS2B3 of dengue virus were developed. The study involves cell transfection studies followed by confocal and single-molecule imaging. The correlative biophysical study (comprising of confocal and single-molecule imaging) and estimated cluster parameters corroborates our findings.

pathology↗

bioRxiv2022

Simultaneous Multisheet Multichannel Imaging Cytometry (SMMIC) System Powered by Adaptive Vertically-Aligned Multi-Sheet Array (VAMSA) PSF

Volumetric interrogating of a large population of live specimens at high throughput is a challenging task that necessitates new technology. We propose vertical-aligned multi-sheet array (VAMSA) illumination PSF that enables interrogation of specimens flowing simultaneously through multiple microfluidic channels. The very geometry of PSF enables high quality cross-sectional imaging, and facilitates volumetric interrogation of specimens flowing through commercial microfluidic chip (consists of multiple flow-channels), which is a step towards large population screening. The SMMIC technique employs a unique combination of transmission grating, beam-expander and high NA objective system in a specific optical configuration to generate diffraction-limited illumination PSF (VAMSA-PSF). However, the detection is accomplished by a large field-of-view widefield 4f-system that consists of low NA objective lens, high performance fluorescence filters, and tube lens. Studies show high quality sectional images (resolution [~] 2.5m, and SBR [~] 4.8dB) of HeLa cancerous cells at high flow throughput (flow-rate of, 2500 nl/min). A cell count of > 1k and volume reconstruction efficiency of [~] 121 cells/min is noteworthy. In addition, SMMIC system demonstrate organelle-level resolution with a SBR comparable to that of confocal especially at low flow-rates. It is hoped that the proposed system may accelerates drug-treatment studies for a large population of live specimens to advance the evolving field of translational medicine and health-care.

biophysics↗

bioRxiv2022

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

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 demonstrated the ability to resolve biological features at the very 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) in the existing SMLM system, volume scanning (along z-axis) 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 clustered fraction) are also determined.

biophysics↗

bioRxiv2022

Detection of Fortunate Molecules Induce PArticle Resolution Shift (PAR-shift) towards Single-molecule Limit in SMLM: A Technique for Resolving Molecular Clusters in Cellular System

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 [Formula]. 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.

biophysics↗

bioRxiv2021

Lightsheet Optical Tweezer (LOT) for Optical Manipulation of Microscopic Particles and Live Cells

A light sheet based optical tweezer (LOT) is developed to trap microscopic dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light sheet which is realized by a combination of cylindrical lens and high NA objective lens. The field pattern generated at the geometrical focus is a tightly focussed line (along x-axis) perpendicular to the beam propagation direction (z-axis). Spherical beads undergoing Brownian motion in the solution are trapped by the gradient potential, and the time (to reach trap-center under the influence of gradient potential) is estimated from the fast CMOS camera (operating at 230frames/sec). High-speed imaging of beads at varying laser power shows a steady increase in the stiffness of LOT with a maximum of 0.00118 pN/nm at 52.5 mW. This is an order less than traditional optical point-traps. The trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting a single degree of freedom. Subsequently, LOT is used to optically trap and pattern dielectric beads and HeLa cells in a line. We could successfully pattern 8 dielectric beads and 3 HeLa cells in a straight line. We anticipate that LOT can be used to study the 1D-physics of microscopic particles and help understand the patterned growth of live cells.

biophysics↗

bioRxiv2021

In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug

The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensin-converting enzyme 2 (ACE2)) leading to its entry in the cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD), and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behavior of S-protein in the presence of these drugs for the first 12 hours post-S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a significant fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD. In contrast, in the presence of Riboflavin, S-protein carries out a normal endocytic pathway, comparable to the control (no drug) group. Therefore, the present study indicates ADD potentially affects S-proteins entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interferes with S-protein cellular entrance mechanism. This is further strengthened by 24 hrs study. However, detailed studies at the molecular scale are necessary to clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveals ADD could be a potential candidate to manage Covid-19 functions through the yet unknown molecular mechanism.

biophysics↗

bioRxiv2020

High Throughput Volume Flow Cytometry (parallel-iLIFE) Resolves Mitochondrial Network On the Go

Cell screening and viability studies are paramount to access cell morphology and intracellular molecular variations within large heterogeneous populations of cells. This forms the basis for diagnosis of infections, evaluating immunohistochemistry and routine histopathology. The proposed volume flow cytometry (also termed as, parallel Integrated Light-sheet imaging and flow-based enquiry (parallel-iLIFE)) is a powerful method that adds new capabilities (3D volume visualization, organelle-level resolution and multi-organelle screening) powered by light sheet based illumination. Unlike state-of-the-art point-illumination based imaging cytometry techniques, light sheet based parallel-iLIFE technique is capable of screening species with high throughput and near diffraction-limited resolution. The flow system was realized on a multichannel (Y-type) microfluidic chip that enables visualization of mitochondrial network of several cells in-parallel at a relatively high flow-rate of 2000 nl/min. The calibration of system requires study of point emitters (fluorescent beads) at physiologically relevant flow-rates (50-2000 nl/min) for determining flow-induced optical aberration in the system point spread function (PSF). Subsequently, recorded raw images and volumes were deconvolved with flow-variant PSF to reconstruct cellular mitochondrial network. High throughput investigation of HeLa cells were carried out at sub-cellular resolution in real-time and critical parameters (mitochondria count and size distribution, morphology and cell strain statistics) are determined on-the-go. These parameters determine the physiological state of cells and the changes in mitochondrial distribution over-time that may have consequences in disease diagnosis. The development of volume flow cytometry system (parallel-iLIFE) and its suitability to study sub-cellular components at high-throughput high-content capacity with organelle-level resolution may enable disease diagnosis on a single microfluidic chip.

bioengineering↗

bioRxiv2020

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

To be able to resolve molecular-clusters it is crucial to access vital informations (such as, molecule density and cluster-size) that are key to understand disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precisions (LPs)) to reconstruct super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single molecules) that degrade image resolution. Ideally it should be possible to identify the brightest molecules (also termed as, the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. POSSIBLE microscopy explores this possibility by introducing narrow probability size-distribution of single molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single molecule filtering is carried out by the Gaussian distribution function to filter out unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single molecule images with an average PSF sizes of{sigma} {+/-} {Delta}{sigma} = 15 {+/-} 10 nm, 30 {+/-} 2 nm & 50 {+/-} 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques.

biophysics↗