Erik Bélanger

In vivo evaluation of demyelination and remyelination in a nerve crush injury model

Nerves of the peripheral nervous system have, to some extent, the ability to regenerate after injury, particularly in instances of crush or contusion injuries. After a controlled crush injury of the rat sciatic nerve, demyelination and remyelination are followed with functional assessments and imaged both ex vivo and in vivo over the course of 4 weeks with video-rate coherent anti-Stokes Raman scattering (CARS) microscopy. A new procedure compatible with live animal imaging is developed for performing histomorphometry of myelinated axons. This allows quantification of demyelination proximal and remyelination distal to the crush site ex vivo and in vivo respectively.

Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams

The use of coherent anti-Stokes Raman scattering microscopy tuned to the lipid vibration for quantitative myelin imaging suffers from the excitation polarization dependence of this third-order nonlinear optical effect. The contrast obtained depends on the orientation of the myelin membrane, which in turn affects the morphometric parameters that can be extracted with image analysis. We show how circularly polarized laser beams can be used to avoid this complication, leading to images free of excitation polarization dependence. The technique promises to be optimal for in vivo imaging and the resulting images can be used for coherent anti-Stokes Raman scattering optical histology on native state tissue.

In vivo optical monitoring of tissue pathologies and diseases with vibrational contrast.

Studies of tissue remodeling require in vivo imaging techniques that are as minimally invasive as possible to avoid microenvironment perturbations. To this end, spontaneous Raman techniques have been used but low signals have limited their application mostly to point spectroscopy measurements. Novel Raman-based techniques such as coherent and stimulated Raman scattering can overcome this limitation. This manuscript discusses imaging and spectroscopy applications with Raman-based contrast for in vivo tissue monitoring, and how these can be combined into spectral imaging.

Live animal myelin histomorphometry of the spinal cord with video-rate multimodal nonlinear microendoscopy

In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.

Highly Efficient and High-Power Raman Fiber Laser Based on Broadband Chirped Fiber Bragg Gratings

A highly efficient and high-power Raman fiber laser was developed based on the use of broadband fiber Bragg gratings (FBGs) as optical couplers. The broadening of the Stokes signal is analyzed in both cases where the laser emission is restricted or not by the FBGs bandwidth. The use of broadband FBGs with minimized cladding-mode losses allows us to overcome the problem of power leakage outside the laser cavity through the input coupler. It is shown that by carefully tailoring the intracavity spectral losses and the FBGs losses, lasing efficiencies approaching the quantum limit can be obtained. In fact, 7.8 W of Stokes power with a conversion efficiency of 93.6% has been obtained

High-Power and Widely Tunable All-Fiber Raman Laser

A high-power and widely tunable all-fiber Raman laser is demonstrated. The Raman fiber laser has been tuned over a range of 60 nm from 1075 to 1135 nm and delivers up to 5.0 W of Stokes output power for 6.5 W of launched pump power. Efficiencies ranging from 76.1 to 93.1% and laser thresholds from 0.78 to 2.59 W have been measured. The spectrum of the depolarized Raman gain coefficient of the germanosilicate fiber has also been inferred from our experimental measurements.

Automated method for the segmentation and morphometry of nerve fibers in large-scale CARS images of spinal cord tissue

A fully automated method for large-scale segmentation of nerve fibers from coherent anti-Stokes Raman scattering (CARS) microscopy images is presented. The method is specifically designed for CARS images of transverse cross sections of nervous tissue but is also suitable for use with standard light microscopy images. After a detailed description of the two-part segmentation algorithm, its accuracy is quantified by comparing the resulting binary images to manually segmented images. We then demonstrate the ability of our method to retrieve morphological data from CARS images of nerve tissue. Finally, we present the segmentation of a large mosaic of CARS images covering more than half the area of a mouse spinal cord cross section and show evidence of clusters of neurons with similar g-ratios throughout the spinal cord.

Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach.

We present an automated two-dimensional Fourier transform (2D-FT) approach to analyze the local organization of myelinated axons in the spinal cord. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to observe lesions in a commonly used animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). A 2D-FT was applied on the CARS images to find the average orientation and directional anisotropy of the fibers within contiguous image domains. We introduce the corrected correlation parameter (CCP), a measure of the correlation between orientations of adjacent domains. We show that in the EAE animal model of MS, the CCP can be used to quantify the degree of organization/disorganization in the myelin structure. This analysis was applied to a large image dataset from animals at different clinical scores and we show that some descriptors of the CCP probability density function are strongly correlated with the clinical scores. This procedure, compatible with live animal imaging, has been developed to perform local in situ evaluation of myelinated axons afflicted by EAE.

Long-term stable device for tuning fiber Bragg gratings

It is demonstrated that with a proper choice of embedding material, the composite beam bending method constitutes an effective and reliable approach for tuning fiber Bragg gratings. A long-term stable device is presented with a dynamic range of 80 nm, which exhibits insertion losses smaller than 0.28 dB and small variations of the full width at half-maximum.

Probing pain pathways with light

We have witnessed an accelerated growth of photonics technologies in recent years to enable not only monitoring the activity of specific neurons, while animals are performing certain types of behavior, but also testing whether specific cells, circuits, and regions are sufficient or necessary for initiating, maintaining, or altering this or that behavior. Compared to other sensory systems, however, such as the visual or olfactory system, photonics applications in pain research are only beginning to emerge. One reason pain studies have lagged behind is that many of the techniques originally developed cannot be directly implemented to study key relay sites within pain pathways, such as the skin, dorsal root ganglia, spinal cord, and brainstem. This is due, in part, to difficulties in accessing these structures with light. Here we review a number of recent advances in design and delivery of light-sensitive molecular probes (sensors and actuators) into pain relay circuits to help decipher their structural and functional organization. We then discuss several challenges that have hampered hardware access to specific structures including light scattering, tissue movement and geometries. We review a number of strategies to circumvent these challenges, by delivering light into, and collecting it from the different key sites to unravel how nociceptive signals are encoded at each level of the neuraxis. We conclude with an outlook on novel imaging modalities for label-free chemical detection and opportunities for multimodal interrogation in vivo. While many challenges remain, these advances offer unprecedented opportunities to bridge cellular approaches with context-relevant behavioral testing, an essential step toward improving translation of basic research findings into clinical applications.