Study of myelin disease by coherent anti-Stokes Raman scattering microscopy

Myelin sheath is a fatty multi-lamellar membrane wrapping around an axon and is crucial for high-speed impulse conduction. Demyelination, loss of normal myelin sheath, accounts for long-term neurologic disability. Multiple sclerosis is the most common inflammatory demyelinating disease affecting more than 2.5 million people worldwide. Focal areas of intense demyelination and white matter infiltration by immune cells are two pathological hallmarks of the disease. The current understanding of the molecular mechanisms underlying myelin damage is very limited, partially due to the lack of microscopic tools for high-resolution imaging of myelin in its natural state. The traditional imaging methods such as electron microscopy, histology, and immunofluorescence focus on the fixed and dehydrated tissues which prevent the dynamic study of cellular and enzymatic activities and in situ study of disease process. Clinical imaging tools including MRI and PET permit in vivo imaging of white matter, but lack sufficient resolutions to reveal details at signal cell level. Here we present a laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy which enables real-time imaging of myelin and demyelination in the fresh tissue and in vivo at the single cell level (Biophys J 2005, 89:581-591). This modality can be combined with sum frequency generation (SFG) and two-photon excitation fluorescence (TPEF) imaging to monitor multiple components including astrocyte processes and calcium in the central nervous system simultaneously (Biophys J 2007, 92:3251-3259). The capability of CARS microscopy was used to map the whole brain white matter in brain slices and to analyze the microstructural anatomy of brain axons. In vivo imaging of subcortex white matter in brain was demonstrated on an upright microscope (Opt Express, 2008, 16:19396-19409). With this advanced system, we have carried out mechanistic study of myelin damage in different demyelination models. Results show that lyso-PtdCho induced-demyelination is a Ca2+ dependent process (J Neurosci Res 2007, 85:2871-2881). Glutamate inflicts paranodal myelin splitting and retraction (PLoS ONE 2008, 4:e6705). Further study has demonstrated that CARS microscopy is an efficient readout of demyelination and remyelination in an animal disease model of multiple sclerosis.