Ex vivo and in vivo coherent Raman imaging of the peripheral and central nervous system.

摘要

A hallmark of nervous system disorders is damage or degradation of the myelin sheath. Unraveling the mechanisms underlying myelin degeneration and repair represent one of the great challenges in medicine. This thesis work details the development and utilization of advanced optical imaging methods to gain insight into the structure and function of myelin in both healthy and diseased states in the in vivo environment.;This first part of this thesis discusses ex vivo studies of the effects of high-frequency stimulation of spinal tissues on the structure of the node of Ranvier as investigated by coherent anti-Stokes Raman scattering (CARS) imaging (manuscript submitted to Journal of Neurosciece). Reversible paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation, beginning minutes after the onset and continuing for up to 10 min after stimulation was ceased. A mechanistic study revealed a Ca2+ dependent pathway: high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down.;Also, the construction of dual-scanning CARS microscope for large area mapping of CNS tissues is detailed (Optics Express, 2008, 16:19396-193409). A confocal scanning head equipped with a rotating polygon mirror provides high speed, high resolution imaging and is coupled with a motorized sample stage to generate high-resolution large-area images of mouse brain coronal section and guinea pig spinal cord cross section. The polygon mirror decreases the mosaic acquisition time significantly without reducing the resolution of individual images.;The ex vivo studies are then extended to in vivo imaging of mouse sciatic nerve tissue by CARS and second harmonic generation (SHG) imaging (Journal of Microscopy, 2007, 225: 175-182). Following a minimally invasive surgery to open the skin, CARS imaging of myelinated axons and SHG imaging of the surrounding collagen fibers were demonstrated with high signal-to-background ratio, 3D spatial resolution, and no need for labeling. The underlying contrast mechanisms of in vivo CARS were explored by 3D imaging of fat cells that surround the nerve. The lessons learned in imaging peripheral nerve were utilized to enable a preliminary study of longitudinal in vivo CARS imaging of myelin degradation and repair. We demonstrate high resolution longitudinal imaging of myelin degradation and remyelination in rat spinal cord by in vivo CARS imaging of the same rats for a period of 4 weeks (manuscript submitted to Nature Methods).;Lastly, two approaches towards achieving greater imaging depth in vivo are discussed. In the first, a miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy (Optics Letters, 2007, 32: 2212-14). By inserting the lens tip into a soft gel sample, CARS images of 2-mum polystyrene beads at 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery. The second study details the demonstration of laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging with two excitation laser beams delivered by a large mode area photonic crystal fiber. The group velocity dispersion and self phase modulation effects are largely suppressed due to the large mode area of the fiber and the use of ps pulses (Optics Letters, 2006, 31:1417-1419).