Quantitative label-free imaging of lipid domains in single bilayers by hyperspectral coherent Raman scattering

Nahmad-Rohen, Alexander ORCID: https://orcid.org/0000-0002-2712-7373, Regan, David ORCID: https://orcid.org/0000-0003-0420-3481, Masia, Francesco ORCID: https://orcid.org/0000-0003-4958-410X, McPhee, Craig, Pope, Iestyn ORCID: https://orcid.org/0000-0002-4104-0389, Langbein, Wolfgang ORCID: https://orcid.org/0000-0001-9786-1023 and Borri, Paola ORCID: https://orcid.org/0000-0002-7873-3314 2020. Quantitative label-free imaging of lipid domains in single bilayers by hyperspectral coherent Raman scattering. Analytical Chemistry 92 (21) 10.1021/acs.analchem.0c03179

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Abstract

Lipid phase separation in cellular membranes is thought to play an important role in many biological functions. This has prompted the development of synthetic membranes to study lipid–lipid interactions in vitro, alongside optical microscopy techniques aimed at directly visualizing phase partitioning. In this context, there is a need to overcome the limitations of fluorescence microscopy, where added fluorophores can significantly perturb lipid packing. Raman-based optical imaging is a promising analytical tool for label-free chemically specific microscopy of lipid bilayers. In this work, we demonstrate the application of hyperspectral coherent Raman scattering microscopy combined with a quantitative unsupervised data analysis methodology developed in-house to visualize lipid partitioning in single planar membrane bilayers exhibiting liquid-ordered and liquid-disordered domains. Two home-built instruments were utilized, featuring coherent anti-Stokes Raman scattering and stimulated Raman scattering modalities. Ternary mixtures of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol were used to form phase-separated domains. We show that domains are consistently resolved, both chemically and spatially, in a completely label-free manner. Quantitative Raman susceptibility spectra of the domains are provided alongside their spatially resolved concentration maps.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Physics and Astronomy Biosciences
Publisher:	American Chemical Society
ISSN:	0003-2700
Funders:	BBSRC, EPSRC
Date of First Compliant Deposit:	13 October 2020
Date of Acceptance:	8 October 2020
Last Modified:	03 Apr 2024 01:07
URI:	https://orca.cardiff.ac.uk/id/eprint/135569

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