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Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

ABSTRACT: Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their movement during mitosis, as well as structures like nucleoli and nuclear border in interphase. Furthermore, the specific organization of the endoplasmic reticulum during mitosis is highlighted. Finally, we stress that MCARS can reveal the biochemical impact of the fixative method at the cellular level. Beyond the study of the cell cycle, this work introduces a label-free imaging approach that enables the visualization of cellular processes where chromatin undergoes rearrangements.

SUBMITTER: Guerenne-Del Ben T

PROVIDER: S-EPMC6761141 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

Guerenne-Del Ben Tiffany T Rajaofara Zakaniaina Z Couderc Vincent V Sol Vincent V Kano Hideaki H Leproux Philippe P Petit Jean-Michel JM

Scientific reports 20190925 1

Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their ...[more]

PMID: 31554897

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Project description:Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Dataset Information

Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

Publications

Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

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