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.
Highlights
Lipid bilayers have been investigated for several decades to study lipid−lipid and lipid−protein interactions.[1]
coherent anti-Stokes Raman scattering (CARS) microspectroscopy was performed with 10 cm−1 resolution via spectral focusing. xyz images were acquired to locate the bilayer in the optimum focal plane, and a series of xy images at wave numbers of 2700−3100 cm−1 was recorded to resolve the lipid vibrational resonances
The CARS intensity ratio relative to the nonresonant CARS background measured in the glass coverslip is shown, with the dispersive lipid resonance being superimposed onto the response from the surround aqueous buffer (PBS)
Summary
Lipid bilayers have been investigated for several decades to study lipid−lipid and lipid−protein interactions.[1] They serve as model systems to aid our understanding of the heterogeneous organization of cellular membranes[2] and in the investigation of many processes, including protein segregation in lipid domains, membrane protein function, drug−receptor interactions, and transmembrane transport. They are receiving increasing attention as building blocks in bottom-up synthetic biology approaches to creating artificial cells.[3]. The quantitative characterization of these heterogeneous lipid domains in single bilayers is not trivial, and the presently utilized techniques have a number of drawbacks
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