Planar lightsheet optical tweezer pLOT for 2D trapping and imaging of freely-moving live cells Neptune Baro, Partha Pratim Mondal Communications Biology, 2025 We report the realization of the first planar optical trap and imaging system using a sheet of light that allows interrogation of living specimens in a plane. An orthogonal widefield detection is employed to directly visualize the trapping of the target object (dielectric beads/cells) in a plane. The planar trap is realized on an inverted optical stage with illumination from the bottom. The system uses a combination of a cylindrical lens and a high NA objective lens to generate a tightly focused diffraction-limited sheet of light. For trapping objects in a selective XZ-plane, the sample (beads/cells suspended in a solution) is illuminated by a sheet of light (along the Z-axis with coverslip along XZ), whereas the detection is carried out perpendicular to the coverslip (along the Y-axis). Orthogonal detection allows direct visualization of the trapped object in the 2D plane illuminated by the lightsheet. The generated PSF has a dimension of 2073.84 μm2 (along XZ), which defines the active trap region/zone. To estimate the trap stiffness, both variance-based equipartition and video-based object tracking methods are employed. Results (image and video) show real-time trapping of dielectric beads and live cells in the trap zone (2D plane). Prolonged exposure shows objects getting trapped and builds up a 2D layer of beads/cells, demonstrating stable trapping in a selective layer. The technique is furthered by successfully trapping fluorescently labeled live cells in a single plane and simultaneously performing fluorescence imaging on the go with sub-cellular resolution. The potential of the planar trap lies in its ability to confine objects (such as dielectric beads and cells) in a selective 2D plane and allow interrogation, thereby opening up the possibility of new kinds of studies in optical manipulation, fluorescence microscopy and biotechnology. Statement of Significance: The ability to confine and interrogate living specimens (cells) in a plane is an incredible feat that paves the way for new kinds of studies. Currently, there is no available technique that can trap microscopic living objects in a 2D plane. The successful trapping of live cells in a plane, and high-resolution fluorescence imaging on the go, have applications in the broad field of fluorescence microscopy, optical physics and biotechnology. Lightsheet based 2D optical tweezer (planar 2DLOT) allows trapping objects (living & non-living) in a 2D plane and simultaneous interrogation/imaging with diffraction-limited resolution.
Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell Jigmi Basumatary, Neptune Baro, Fancesca Cella Zanacchi, Partha Pratim Mondal Scientific Reports, 2023 The blinking properties of a single molecule are critical for single-molecule localization microscopy (SMLM). Typically, SMLM techniques involve recording several frames of diffraction-limited bright spots of single-molecules with a detector exposure time close to the blinking period. This sets a limit on the temporal resolution of SMLM to a few tens of milliseconds. Realizing that a substantial fraction of single molecules emit photons for time scales much shorter than the average blinking period, we propose accelerating data collection to capture these fast emitters. Here, we put forward a short exposure-based SMLM (shortSMLM) method powered by sCMOS detector for understanding dynamical events (both at single molecule and ensemble level). The technique is demonstrated on an Influenza-A disease model, where NIH3T3 cells (both fixed and live cells) were transfected by Dendra2-HA plasmid DNA. Analysis shows a 2.76-fold improvement in the temporal resolution that comes with a sacrifice in spatial resolution, and a particle resolution shift PAR-shift (in terms of localization precision) of $$\\sim$$ ∼ 11.82 nm compared to standard SMLM. We visualized dynamic HA cluster formation in transfected cells post 24 h of DNA transfection. It is noted that a reduction in spatial resolution does not substantially alter cluster characteristics (cluster density, $$\\#$$ # molecules/cluster, cluster spread, etc.) and, indeed, preserves critical features. Moreover, the time-lapse imaging reveals the dynamic formation and migration of Hemagglutinin (HA) clusters in a live cell. This suggests that $$short-SMLM$$ s h o r t - S M L M using a synchronized high QE sCMOS detector (operated at short exposure times) is excellent for studying temporal dynamics in cellular system.
Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging Jigmi Basumatary, Neptune Baro, Prakash Joshi, Partha Pratim Mondal Communications Biology, 2023 Over the last decade, single-molecule localization microscopy (SMLM) has developed into a set of powerful techniques that have improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features down to a few tens of nanometers. We introduce a single molecule-based scanning SMLM ( s c a n S M L M ) system that enables rapid volume imaging. Along with epi-illumination, the system employs a scanning-based 4f detection for volume imaging. The 4f system comprises a combination of an electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of an electrically-tunable lens (ETL) in a 4f detection system, the selectivity of the axial object plane is achieved, for which the image forms in the image/detector plane. So, in principle, one can scan the object volume by just altering the aperture of ETL. Two schemes were adopted to carry out volume imaging: cyclic scan and conventional scan. The cyclic scheme scans the volume in each scan cycle, whereas plane-wise scanning is performed in the conventional scheme. Hence, the cyclic scan ensures uniform dwell time on each frame during data collection, thereby evenly distributing photobleaching throughout the cell volume. With a minimal change in the system hardware (requiring the addition of an ETL lens and related electronics for step-voltage generation) in the existing SMLM system, volume scanning (along the z-axis) can be achieved. To calibrate and derive critical system parameters, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, s c a n S M L M is employed to visualize the architecture of actin-filaments and the distribution of Meos-Tom20 molecules on the mitochondrial membrane. The technique is further exploited to understand the clustering of Hemagglutinin (HA) protein single molecules in a transfected cell for studying Influenza-A disease progression. The system, for the first time, enabled 3D visualization of HA distribution that revealed HA cluster formation spanning the entire cell volume, post 24 hrs of transfection. Critical biophysical parameters related to HA clusters (density, the number of HA molecules per cluster, axial span, fraction of clustered molecules, and others) are also determined, giving an unprecedented insight into Influenza-A disease progression at the single-molecule level.
Lightsheet optical tweezer (LOT) for optical manipulation of microscopic particles and live cells Partha Pratim Mondal, Neptune Baro, Ankur Singh, Prakash Joshi, Jigmi Basumatary Scientific Reports, 2022 Optical trapping and patterning cells or microscopic particles is fascinating. We developed a light sheet-based optical tweezer to trap dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light-sheet realized by a combination of cylindrical lens and high NA objective lens. The resultant field is a focussed line (along x -axis) perpendicular to the beam propagation direction ( z -axis). This is unlike traditional optical tweezers that are fundamentally point-traps and can trap one particle at a time. Several spherical beads undergoing Brownian motion in the solution are trapped by the lightsheet gradient potential, and the time (to reach trap-centre) is estimated from the video captured at 230 frames/s. High-speed imaging of beads with increasing laser power shows a steady increase in trap stiffness with a maximum of 0.00118 pN/nm at 52.5 mW. This is order less than the traditional point-traps, and hence may be suitable for applications requiring delicate optical forces. On the brighter side, light sheet tweezer (LOT) can simultaneously trap multiple objects with the distinct ability to manipulate them in the transverse ( xy ) plane via translation and rotation. However, the trapped beads displayed free movement along the light-sheet axis ( x -axis), exhibiting a single degree of freedom. Furthermore, the tweezer is used to trap and pattern live HeLa cells in various shapes and structures. Subsequently, the cells were cultured for a prolonged period of time (> 18 h), and cell viability was ascertained. We anticipate that LOT can be used to study constrained dynamics of microscopic particles and help understand the patterned cell growth that has implications in optical imaging, microscopy, and cell biology.
RECENT SCHOLAR PUBLICATIONS
Planar lightsheet optical tweezer pLOT for 2D trapping and imaging of freely-moving live cells N Baro, PP Mondal Communications Biology 8 (1), 1763 , 2025 2025
LIGHT-SHEET OPTICAL TWEEZER PP Mondal, N Baro IN Patent 544,480 , 2024 2024
TRapping and IMaging (TRIMing) of Cells/Multicellular Organisms in Free Living Environment Enabled by Adaptive Lightsheet Optical Tweezer (aLOT) N Baro, J Basumatary, N Pant, PP Mondal bioRxiv, 2024.05. 02.591710 , 2024 2024 Citations: 2
Planar Optical Tweezer Trap (2D-LOT) System Realized by Light Sheet Illumination & Orthogonal Widefield Detection N Baro, PP Mondal bioRxiv, 2024.04. 15.589441 , 2024 2024 Citations: 1
Realization of Planar Optical Tweezer (2D-LOT) Powered by Light Sheet N Baro, PP Mondal arXiv preprint arXiv:2404.10265 , 2024 2024
Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging J Basumatary, N Baro, P Joshi, PP Mondal Communications Biology 6 (1), 1050 , 2023 2023 Citations: 13
Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell J Basumatary, N Baro, FC Zanacchi, PP Mondal Scientific Reports 13 (1), 12561 , 2023 2023 Citations: 6
Lightsheet optical tweezer (LOT) for optical manipulation of microscopic particles and live cells PP Mondal, N Baro, A Singh, P Joshi, J Basumatary Scientific Reports 12 (1), 10229 , 2022 2022 Citations: 38
Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging J Basumatary, N Baro, P Joshi, PP Mondal bioRxiv, 2022.04. 01.486682 , 2022 2022
MOST CITED SCHOLAR PUBLICATIONS
Lightsheet optical tweezer (LOT) for optical manipulation of microscopic particles and live cells PP Mondal, N Baro, A Singh, P Joshi, J Basumatary Scientific Reports 12 (1), 10229 , 2022 2022 Citations: 38
Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging J Basumatary, N Baro, P Joshi, PP Mondal Communications Biology 6 (1), 1050 , 2023 2023 Citations: 13
Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell J Basumatary, N Baro, FC Zanacchi, PP Mondal Scientific Reports 13 (1), 12561 , 2023 2023 Citations: 6
TRapping and IMaging (TRIMing) of Cells/Multicellular Organisms in Free Living Environment Enabled by Adaptive Lightsheet Optical Tweezer (aLOT) N Baro, J Basumatary, N Pant, PP Mondal bioRxiv, 2024.05. 02.591710 , 2024 2024 Citations: 2
Planar Optical Tweezer Trap (2D-LOT) System Realized by Light Sheet Illumination & Orthogonal Widefield Detection N Baro, PP Mondal bioRxiv, 2024.04. 15.589441 , 2024 2024 Citations: 1
Planar lightsheet optical tweezer pLOT for 2D trapping and imaging of freely-moving live cells N Baro, PP Mondal Communications Biology 8 (1), 1763 , 2025 2025
LIGHT-SHEET OPTICAL TWEEZER PP Mondal, N Baro IN Patent 544,480 , 2024 2024
Realization of Planar Optical Tweezer (2D-LOT) Powered by Light Sheet N Baro, PP Mondal arXiv preprint arXiv:2404.10265 , 2024 2024
Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging J Basumatary, N Baro, P Joshi, PP Mondal bioRxiv, 2022.04. 01.486682 , 2022 2022
