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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↗