Biology subjects

Mondal, P. P.

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

4 recordsLinked to original sources

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↗