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Hyperspectral volumetric coherent anti-Stokes Raman scattering microscopy: quantitative volume determination and NaCl as non-resonant standard.

ABSTRACT: In this work, we demonstrate quantitative volume determination of chemical components in three dimensions using hyperspectral coherent anti-Stokes Raman scattering microscopy, phase-corrected Kramers-Kronig retrieval of the coherent anti-Stokes Raman scattering susceptibility and factorization into concentration of chemical components. We investigate the influence of the refractive index contrast between water and polymer beads (polystyrene and polymethylmethacrylate), showing that it leads mainly to concentration errors, while the spectral error is less affected. The volume of polystyrene beads of sizes from 200 nm to 3 μm is determined with 10% relative error and 1% absolute error in the region of interest. We furthermore establish the use of sodium chloride as non-resonant reference material free of Raman-active vibrational resonances.

SUBMITTER: Karuna A

PROVIDER: S-EPMC5032898 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other

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Hyperspectral volumetric coherent anti-Stokes Raman scattering microscopy: quantitative volume determination and NaCl as non-resonant standard.

Karuna Arnica A Masia Francesco F Borri Paola P Langbein Wolfgang W

Journal of Raman spectroscopy : JRS 20160210 9

In this work, we demonstrate quantitative volume determination of chemical components in three dimensions using hyperspectral coherent anti-Stokes Raman scattering microscopy, phase-corrected Kramers-Kronig retrieval of the coherent anti-Stokes Raman scattering susceptibility and factorization into concentration of chemical components. We investigate the influence of the refractive index contrast between water and polymer beads (polystyrene and polymethylmethacrylate), showing that it leads main ...[more]

PMID: 27708499

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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

Hyperspectral volumetric coherent anti-Stokes Raman scattering microscopy: quantitative volume determination and NaCl as non-resonant standard.

Publications

Hyperspectral volumetric coherent anti-Stokes Raman scattering microscopy: quantitative volume determination and NaCl as non-resonant standard.

Similar Datasets

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