Biology subjects

Basumatary, J.

Publications and source records attributed to Basumatary, J..

5 recordsLinked to original sources

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↗

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↗

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↗

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↗