Biology subjects

Baro, N.

Publications and source records attributed to Baro, N..

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↗

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↗

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↗

bioRxiv2021

The world's hotspot of linguistic and biocultural diversity under threat

Papua New Guinea is home to >10% of the worlds languages and rich and varied biocultural knowledge, but the future of this diversity remains unclear. We measured language skills of 6,190 students speaking 392 languages (5.5% of the global total) and modelled their future trends, using individual-level variables characterizing family language use, socio-economic conditions, students skills, and language traits. This approach showed that only 58% of the students, compared to 91% of their parents, were fluent in indigenous languages, while the trends in key drivers of language skills (language use at home, proportion of mixed-language families, urbanization, students traditional skills) predicted accelerating decline of fluency, to an estimated 26% in the next generation of students. Ethnobiological knowledge declined in close parallel with language skills. Varied medicinal plant uses known to the students speaking indigenous languages are replaced by a few, mostly non-native species for the students speaking English or Tok Pisin, the national lingua franca. Most (88%) students want to teach indigenous language to their children. While crucial for keeping languages alive, this intention faces powerful external pressures as key factors (education, cash economy, road networks, urbanization) associated with language attrition are valued in contemporary society. Significance StatementAround the world, more than 7,000 languages are spoken, most of them by small populations of speakers in the tropics. Globalization puts small languages at a disadvantage, but our understanding of the drivers and rate of language loss remains incomplete. When we tested key factors causing language attrition among Papua New Guinean students speaking 392 different indigenous languages, we found an unexpectedly rapid decline in their language skills compared to their parents and predicted further acceleration of language loss in the next generation. Language attrition was accompanied by decline in the traditional knowledge of nature among the students, pointing to an uncertain future for languages and biocultural knowledge in the most linguistically diverse place on Earth.

ecology↗