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Mammalian cell and tissue imaging using Raman and coherent Raman microscopy

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Abstract Direct imaging of metabolism in cells or multicellular organisms is important for understanding many biological processes. Raman scattering (RS) microscopy, particularly, coherent Raman scattering (CRS) such as coherent anti‐Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has emerged as a powerful platform for cellular imaging due to its high chemical selectivity, sensitivity, and imaging speed. RS microscopy has been extensively used for the identification of subcellular structures, metabolic observation, and phenotypic characterization. Conjugating RS modalities with other techniques such as fluorescence or infrared (IR) spectroscopy, flow cytometry, and RNA‐sequencing can further extend the applications of RS imaging in microbiology, system biology, neurology, tumor biology and more. Here we overview RS modalities and techniques for mammalian cell and tissue imaging, with a focus on the advances and applications of CARS and SRS microscopy, for a better understanding of the metabolism and dynamics of lipids, protein, glucose, and nucleic acids in mammalian cells and tissues. This article is categorized under: Laboratory Methods and Technologies > Imaging Biological Mechanisms > Metabolism Analytical and Computational Methods > Analytical Methods
(a) A diagram showing visible monochromatic laser light scattered by a molecule. Inelastic scattering can be recorded and displayed on a spectrum. (b) An example spectrum of observed Raman scattering with arbitrary intensity units on the vertical axis and Raman shifts in wavenumbers or centimeters inversed on the horizontal axis, displaying the relative incidence of scattered light of certain wavelength and therefore bond vibrations in the sample molecule (Kumamoto, Harada, Takamatsu, & Tanaka, 2018). (c) An electronic energy diagram depicting the difference between Rayleigh scattering (filtered out of Raman systems), RS in green, and electronic pre‐resonance (EPR) in red, and resonant Raman in blue (Reprinted from (Auner et al., 2018) under the terms of the Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/). (d) An illustration of a stimulated Raman system including a Stokes beam to make inelastically scattered light coherent to aid detection and boost intensity. (e) Energy diagram and basis of how a stimulated system's laser source is comprised of a Pump and Stokes beam overlaid in space and time to create a stimulated Raman gain/loss and augment the intensity of a Raman signal is shown (Adapted with permission from Hill and Fu (2019). Copyright 2019 American Chemical Society)
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Application of SRS imaging in mammalian cells. (a) MCF‐7 cells cultured in mediums with metabolic labels (Zhang & Min, 2017, 2020). (b) Spectral unmixing of D‐labeled lipids, proteins, and DNA in Cos7 cells with DO‐SRS (Adapted with permission from Shi, Zheng, et al. (2018). Copyright 2018 Springer Nature). (c) D9‐choline‐containing metabolites in different cancer and embryonic cell lines (Reproduced with permission from Ji et al. (2018). Copyright 2018 The Royal Society of Chemistry). (d) SRS images reveal protein degradation kinetics in HeLa cells (Reproduced with permission from Shen, Xu, Wei, Hu, and Min (2014). Copyright 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim). (e) Multiplexed imaging of macromolecule biosynthesis activity using STRIDE of [D7]‐glucose. Images of a [D7]‐glucose‐labeled mitotic HeLa cell before and after unmixing (Zhang et al., 2019). (f) Label‐free SRS imaging of DNA (magenta), protein (blue), and lipids (green) in live cells in mitotic phase and interphase (Adapted with permission from Lu et al. (2015). Copyright 2015 National Academy of Sciences
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Application of CARS imaging in mammalian cells. (a) Imaging reveals strong glucose import in lipid‐rich cells (left) and lipid‐poor cells (right) (Adapted with permission from Le and Cheng (2009) under the terms of the Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/). (b) Lipid‐droplet composition and packing dependent on incubation medium (Reprinted with permission from Rinia, Burger, Bonn, and Müller (2008). Copyright 2008 The Biophysical Society. Published by Elsevier Inc. All rights reserved). (c) Imaging of L929 cell nuclei (Adapted with permission from Parekh, Lee, Aamer, and Cicerone (2010). Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved). (d) Analysis of interphase and mitotic, stained, fixed and living, HEK293 cells and fibroblasts (Adapted with permission from Guerenne‐Del Ben et al. (2019) under the terms of the Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/)
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Deuterium as a stable‐isotope Raman tag. (a) Proposed mechanisms of incorporation of Deuterium (Left) and how this incorporation manifests in the cell‐silent region (Right). (b) Normalized CDL CDP and CDDNA signals (Left) used to unmix SRS images (Right). Cell‐silent region signals appear as viable as the signals used from the C‐H stretching region. (c) SRS images of various tissues after enzymatic incorporation of a deuterium from heavy water in animal feed (Adapted with permission from Shi, Zheng, et al. (2018). Copyright 2018 Springer Nature)
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Stimulated Raman imaging of cleared tissue. (a–f) Label‐free protein and lipid channels of a 3D MCF‐10AHras tumor spheroid overlapped and independently imaged at various depths with and without clearing. (g) Through‐skull imaging of a glioblastoma with (h) tumor boundaries clearly shown. (i) Computational histology of the tumor/brain interface using protein and lipid channels of the white boxed regions in (g) at specified imaging depths. (Scale Bars: 1 mm (g,h and j), 50 µm (I‐left), 20 µm (I‐right)). (j) Protein/lipid ratiometric image at 100 µm imaging depth. (a–j, Adapted with permission from Wei et al. (2019). Copyright 2019 National Academy of Sciences)
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Analytical and Computational Methods > Analytical Methods
Biological Mechanisms > Metabolism
Laboratory Methods and Technologies > Imaging

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