3/31/2023 0 Comments Index of live home 3d![]() ![]() This coherent noise perturbs the quality of the generated holograms and impedes spatial resolution, which results in reduced RI sensitivity and perceived image quality. While RI distributions have begun to be used in life sciences studies and specific RI signatures for cell structures have been partially analyzed, HTM systems can suffer from coherent noise created by scattered light from the rotational scanning mechanism. Importantly, dynamically adjustable mirrors allow the optics of the HTM device to self-adjust during an acquisition experiment in order to adapt to sample changes such as medium evaporation. The synthesis of the rotational series of scattering spectra is achieved using complex field deconvolution in order to reconstruct a full 3D refractive index (RI) tomogram of even live samples. ![]() Moreover, the device combines this classical holographic approach with rotational scanning of the specimen using a rotating arm equipped with a mirror as described in Fig 1A. The two beams are later brought to interference, and the resulting hologram is recorded on a complementary metal oxide semiconductor (CMOS) camera. A partially coherent light beam generated by a laser diode (520 nm) is split into two beams to create a Mach–Zehnder interferometer setup the first one, called the object beam, interacts with the sample before being collected by a 60× objective, while the second one is the reference beam. The HTM device used in this study is based on quantitative phase microscopy, in which the object’s complex wave field is encoded into a hologram. In this context, holo-tomographic microscopy (HTM) is of great interest because it can provide label-free, high-content images using a very low-power light source that generates no phototoxicity. Classical label-free imaging techniques, while less perturbing, provide images with low information content because of poor contrast and resolution. Photobleaching, phototoxicity, and interference of markers or ectopically expressed engineered proteins are major concerns. Each of these techniques comes with limitations. ![]() Human retinal pigment epithelial cell line SIFT,īecause of the transparent nature of a cell, microscopy techniques either use fluorescent markers or transform optical properties of the sample into an observable contrast (for example, phase contrast, differential interference contrast ). École polytechnique fédérale de Lausanne ER, Mathieu Fréchin is an employee of Nanolive SA.Ĭoherent Anti-Stokes Raman Scattering CMOS,Ĭomplementary metal oxide semiconductor CP3,ĭifferential interference contrast EPFL, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: I have read the journal's policy, and the authors of this manuscript have the following competing interests: Dr. PS, CT, and FGvdG were funded by LipidX and SNSF. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by a grant from the Swiss National Science Foundation (SNSF) and the Swiss SystemsX.ch initiative evaluated by the Swiss National Science Foundation (LipidX). Received: SeptemAccepted: NovemPublished: December 19, 2019Ĭopyright: © 2019 Sandoz et al. Schmid, UT Southwestern Medical Center, UNITED STATES PLoS Biol 17(12):Īcademic Editor: Sandra L. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.Ĭitation: Sandoz PA, Tremblay C, van der Goot FG, Frechin M (2019) Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux. ![]() We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells’ division that have so far, to the best of our knowledge, been out of reach. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell’s refractive indices (RIs) in three dimensions at high spatial and temporal resolution. ![]()
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