Optical sectioning enables 3D bioimaging, but it requires non-optical techniques, such as sample adhesion and mechanical scanning, to hold and manipulate cells. In situ living cells may lack mechanical attachment or support, and may experience stress from artificial adhesion.
A non-contact solution for optical sectioning could broaden the use of 3D imaging to include live cells suspended in high-fluidity environments, such as water or air. Extending optical sectioning to these nonadherent targets is essential for bioimaging cellular structure and dynamics.
Researchers at the Xi’an Institute of Optics and Precision Mechanics (XIOPM) of the Chinese Academy of Sciences, working with a team at the Swiss Federal Institute of Technology, Lausanne (EPFL), developed a method to visualize suspended cells in 3D. Their approach couples structured illumination microscopy (SIM) with holographic optical tweezers. The holographic tweezers enable multiple cells to be manipulated simultaneously using customized structured light.
The developed method, called optical tweeze-sectioning microscopy (OTSM), uses optical processes for both cell immobilization and axial scanning, eliminating the need to affix the samples. OSTM acquires three-step phase-shifting images at each slice of the sample and reconstructs the slices into optical sectioning 3D images. It is an all-optical method and achieves sample scanning through optical delivery of the cells, instead of through translation stages.
To demonstrate OTSM, the researchers used an array of optical traps to capture multiple suspended yeast cells. OTSM enabled precise geometric trapping of 12 suspended live yeast cells into hexagonal, pentagonal, and ring shapes.
To alleviate the risk of photodamage, the researchers used a biocompatible near-infrared wavelength (1064 nm) for the optical traps. They used petal-like traps with a wider lateral dimension than standard Gaussian traps, which reduced the power density experienced by the cells. There was no observable damage to the cells during the experiment, even at the highest power (100 mW).
The team showed that OTSM could achieve full-volume imaging by using axial scanning to capture three-step phase-shifted images at each depth. The holographic optical trapping method trapped cells within structured illumination stripe periods, significantly reducing motion blur and ensuring stable axial scanning. SIM reconstruction produced high-resolution slices, enabling contact-free, high-fidelity 3D image reconstruction. The reconstructed images revealed distinct cellular features with dark shells enclosing bright cores.
The researchers developed a formula to quantify the effect of residual stripes in the reconstructed images — meeting a challenge specific to SIM-based optical scanning. They demonstrated that the effect could be minimized by preprocessing raw images with a background filter.
They showed that the position fluctuations of the cells could be optically squeezed to tens of nm, which is sufficient to implement optical scanning with SIM. Holographic optical trapping suppressed the motion of the suspended cells so that their positional fluctuations were smaller than the imaging resolution and the stripe period of structured illumination, which is essential for SIM.
The OTSM microscopy method enables assembly with controllable distances between cells and the imaging of multiple desired targets, while excluding undesired ones, offering a versatile platform for studying intercellular interaction and biomechanics.
OTSM technology overcomes the limitations of conventional bioimaging techniques that rely on static samples and mechanical scanning. “It promotes the integration of structured illumination microscopy and optical manipulation, and the cross-disciplinary fusion of optical tweezers with other imaging techniques to meet the demands for isotropic resolution, large field of view, and superresolution imaging,” professor Baoli Yao said.
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