Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms

Baro

Singh

et al. 2022

Sci Rep

Self Cite

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.

Section: Resultsmentioning

confidence: 99%

Lightsheet optical tweezer (LOT) for optical manipulation of microscopic particles and live cells

Baro

Singh

et al. 2022

Sci Rep

Self Cite

A perspective on light sheet microscopy and imaging: Applications across the breadth of applied physics and biophysics

Applied Physics Letters

2021

Once in a while, a field of research comes across a technological breakthrough that drives it forward. This is something that seems apparent in optical fluorescence microscopy and imaging with the advent of light sheet technology. Over a span of a few years, this technology has revolutionized the field and has brought in the concept of sheet-based plane-selective imaging with near-diffraction-limited resolution. The technology has progressed further by its integration with existing imaging methods and super-resolution techniques. Unlike existing techniques that are predominantly point-illumination-based, light sheet technology offers selectivity, single-shot interrogation, a large field-of-view, and high-speed data acquisition. Qualitatively, this has resulted in an improved signal-to-background ratio, reduced scattering, improved contrast, and the ability to interrogate a large spectrum of specimens (from cells to mammals). Recent advances have shown the reach of this emerging technology in biological sciences (developmental biology and imaging flow cytometry) and applied physics (nanolithography, beam shaping, and optical traps). The growth and adaptability of this technique are well received by the research community. The technique is expected to grow at a steady rate and has the ability to overcome the issues faced by the existing point-based techniques. In this perspective, we look at the challenges of live imaging (for mammals and plants), the methods in applied physics, and the opportunities presented by recent advances.

Field dipole interaction and polarization effects in light-sheet optical fluorescence microscopy

Kumar

Journal of Applied Physics

2023

Polarization plays a crucial role in understanding the interaction of fluorescent molecules in a light field. We report the study on the effect of a field–dipole interaction under polarization light-sheet fluorescence microscopy using the vectorial theory of light. The molecule is suitably modeled as a radiating electric dipole in a polarized electric field (both linear and random), and the system point spread function (PSF) is determined for different orientations of the dipole (both fixed and random). PSF analysis and contour plots suggest distinct nature of a field distribution in each case, indicating the importance of a field–dipole interaction for high-quality fluorescence imaging. The analysis suggests that the field spreads gradually along the polarization axis at a high numerical aperture (NA) of the objective lens, whereas it is more isotropic and homogeneous at low NA. Moreover, fast changes are not observed at low NA (i.e., far from the central lobe in the field contour plots), suggesting the absence of high-frequency components. However, sidelobes are prominent for linear polarized (along x) light. On the other hand, rapid variations are evident for randomly polarized light, depicting the presence of high spatial frequencies in the system optical transfer function. The other significant observation is the distinct frequency spectrum (both kx and ky) for random and fixed dipoles, indicating the significance of dipole orientation in a light-sheet field. Compared to the point-illumination-based fluorescence microscopy, sheet based polarization technique provides a high signal-to-noise ratio, a uniform field, an order large field of view, and critical information (related to the micro-environment of a dipole and its short-range interactions). The study is expected to facilitate polarization-sensitive investigation of large biological specimens (both fixed and live).