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.

Optical imaging is paramount for disease diagnosis and to access its progression over time. The proposed optical flow imaging (VFC/iLIFE) is a powerful technique that adds new capabilities (3D volume visualization, organelle-level resolution, and multi-organelle screening) to the existing system. Unlike state-of-the-art point-illumination-based biomedical imaging techniques, the sheet-based VFC technique is capable of single-shot sectional visualization, high throughput interrogation, real-time parameter estimation, and instant volume reconstruction with organelle-level resolution of live specimens. The specimen flow system was realized on a multichannel (Y-type) microfluidic chip that enables visualization of organelle distribution in several cells in-parallel at a relatively high flow-rate (2000 nl/min). The calibration of VFC system requires the study of point emitters (fluorescent beads) at physiologically relevant flow-rates (500–2000 nl/min) for determining flow-induced optical aberration in the system point spread function (PSF). Subsequently, the recorded raw images and volumes were computationally deconvolved with flow-variant PSF to reconstruct the cell volume. High throughput investigation of the mitochondrial network in HeLa cancer cell was carried out at sub-cellular resolution in real-time and critical parameters (mitochondria count and size distribution, morphology, entropy, and cell strain statistics) were determined on-the-go. These parameters determine the physiological state of cells, and the changes over-time, revealing the metastatic progression of diseases. Overall, the developed VFC system enables real-time monitoring of sub-cellular organelle organization at a high-throughput with high-content capacity.

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 has demonstrated the ability to resolve biological features at 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 a minimal change in the system hardware (requiring addition of ETL lens and related hardware for step-voltage generation and time synchronization) in the existing SMLM system, volume scanning 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 the 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 3D cells that reveal its clustering across the cell volume. Critical biophysical parameters related to HA clusters (density, #HA/cluster and cluster fraction) are also determined for a single NIH3T3 cell transfected with photoactivable Dendra2-HA plasmid DNA.

Continuous monitoring of large specimens for long durations requires fast volume imaging. This is essential for understanding the processes occurring during the developmental stages of multicellular organisms. One of the key obstacles of fluorescence based prolonged monitoring and data collection is photobleaching. To capture the biological processes and simultaneously overcome the effect of bleaching, we developed single- and multi-color lightsheet based OVSS imaging technique that enables rapid screening of multiple tissues in an organism. Our approach based on OVSS imaging employs quantized step rotation of the specimen to record 2D angular data that reduces data acquisition time when compared to the existing light sheet imaging system (SPIM). A co-planar multicolor light sheet PSF is introduced to illuminate the tissues labelled with spectrally-separated fluorescent probes. The detection is carried out using a dual-channel sub-system that can simultaneously record spectrally separate volume stacks of the target organ. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged the Drosophila larvae and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish for a prolonged duration (5 days) were carried out to understand muscle structuring (Dystrophin, microfibers), primitive Macrophages (in yolk-sac) and inter-dependent lipid and protein-based metabolism. The volume-based study, intensity line-plots and inter-dependence ratio analysis allowed us to understand the transition from lipid-based metabolism to protein-based metabolism during early development (Pharyngula period with a critical transition time, $$\tau _c = 50$$ τ c = 50 h post-fertilization) in Zebrafish. The advantage of multicolor lightsheet illumination, fast volume scanning, simultaneous visualization of multiple organs and an order-less photobleaching makes OVSS imaging the system of choice for rapid monitoring and real-time assessment of macroscopic biological organisms with microscopic resolution.

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 were trapped by the gradient potential and the travel time is estimated from the fast CMOS camera (operating at 230 frames / sec). High-speed imaging of beads shows the stiffness of LOT system to be ≈ 0.00118 ~pN/nm, which is an order less than that of traditional optical point-traps. The trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting one degree of freedom. Subsequently, LOT technique 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.