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.
Analytical and finite element analysis modeling methods of the pulsed-laser excited photothermal (PT) lens signal of solids samples surrounded by air are presented. The analytical and finite element analysis solutions for the temperatures induced in the sample and in the air were found to agree over the range of conditions in this report. Model results show that the air contribution to the total PT lens signal is significant in many cases. In fact, these solutions open up the possibility of applying the pulsed excited thermal lens method for accurate prediction of the heat transfer to the coupling fluid and subsequently to study the gas surrounding the samples by using a known material solid sample.
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.
This paper presents an improved theoretical description of the mode-mismatched thermal lens effect using models that account for heat transport both within the sample and out to the surrounding coupling medium. Analytical and numerical finite element analysis (FEA) solutions are compared and subsequently used to model the thermal lens effect that would be observed using continuous laser excitation. FEA model results were found to be in excellent agreement with the analytical solutions. The model results show that heat transfer to the air coupling medium introduces only a minor effect when compared with the solution obtained without considering axial air-sample heat flux for practical examples. On the other hand, the thermal lens created in the air coupling fluid has a relatively more significant effect on the time-dependent photothermal lens signals.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.