Lipid Order Degradation in Autoimmune Demyelination Probed by Polarized Coherent Raman Microscopy

Abstract

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.

Label‐free non‐linear microscopy to measure myelin outcome in a rodent model of Charcot‐Marie‐Tooth diseases

“…Using CARS imaging, the myelin sheath is directly labelled thank to its rich content in lipids and it appears as a large band surrounding axons. These results are consistent with previous analysis by specialists in optics and in peripheral nerve biology .…”

“…Another interesting improvement is the use of rotating polarization CARS to detect the spatial orientation of CH 2 ‐moieties on lipids . In “healthy” myelin moieties, polarity is much more homogenous than in diseased or aging myelin . In conclusion, non‐linear label‐free CARS microscopy and potentially endoscopy represents a consistent and strong readout tool in the development of new treatments for demyelinating peripheral neuropathies .…”

“…This approach is termed coherent anti‐stokes Raman scattering (CARS) microscopy. While CARS microscopy can be used to image different kinds of molecules, it is particularly efficient in detecting lipids and lipid‐enriched myelin has been one of the first biological objects to be imaged by physicists using CARS .…”

“…These different non‐linear optical microscopy techniques have the important advantage of being label‐free and are therefore applicable for in vivo imaging. However, SHG, THG and CARS techniques are not widely used in the biological and medical communities because the experimental apparatus of the excitation laser and the microscope are complicated, costly and require spatial and temporal synchronization between 2 independent laser sources . We recently addressed this problem by adapting a commercial setup involving a single laser source duplicated through an optical parametric oscillator (OPO) and by temporally synchronizing the 2 laser lines through a delay line .…”

Discriminating polymorph distributions in pharmaceutical tablets using stimulated Raman scattering microscopy

“…Over the last decades, coherent Raman imaging techniques such as coherent anti‐Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have emerged, taking advantage of the high (×10 6 ) enhancement due to the coherent addition of the vibrational emitters . These techniques have spread over many fields to image cells, tissues, plants, cosmetics, nanoparticle drugs, and pharmaceutical compounds . However, only one study has been reported on polymorph imaging and identification using CARS .…”