Despite the interest in studying and quantifying the structural integrity of myelin in postmortem brain tissue, current methods for high-resolution imaging of myelin with optical microscopy are not sufficient. While imaging methods must have adequate resolution and sensitivity to detect microstructural alterations to myelin that are relevant in aging and neurodegenerative disease, an equally critical aspect is to minimize myelin damage that is induced during tissue processing steps. Birefringence microscopy (BRM) is a powerful technique that leverages the structural anisotropy of myelin to provide detailed, label-free images of myelin at any diffraction-limited optical resolution, while maintaining a simple and low-cost setup. Building on our previous work, we have developed a new BRM system and image processing pipeline that enable efficient, high-throughput imaging of myelin structure at multiple scales. Here, we utilize this system to systematically assess the damage to myelin that is induced by several common tissue processing steps in brain sections from the rhesus monkey. Images taken of the same myelinated axons, before and after each tissue processing step, provide direct evidence that mishandling of tissue during sample preparation can cause significant structural alterations to myelin. First, we report on key advancements to our BRM system, imaging procedure, and image processing pipeline, which provide significant increases to the speed and efficiency of BRM. These include integrating fast piezoelectric rotational stages, minimizing the number of images required (to three images) for determining birefringence parameter maps, and implementing an analytical solution for directly determining birefringence parameter maps. Second, using this BRM system, we demonstrate that effective myelin imaging requires (1) the avoidance of prolonged drying or dehydration of tissue, (2) the selection of the optimal mounting medium (85% glycerol), (3) the avoidance of tissue permeabilization with detergents (i.e., Triton X-100 and Saponin), and (4) the selection of a suitable tissue-section thickness (15, 30 and 60 µm) based on the region of interest. In addition to serving as a guide for new users interested in imaging myelin, these basic experiments in sample preparation highlight that BRM is very sensitive to changes in the underlying lipid structure of myelin and suggest that optimized BRM can enable new studies of myelin breakdown in disease. In this work, we show that BRM is a leading method for detailed imaging and characterization of myelin, and we provide direct evidence that the structure of myelin is highly sensitive to damage during inadequate preparation of brain tissue for imaging, which has previously not been properly characterized for birefringence imaging of myelin. For the most effective, high-resolution imaging of myelin structure, tissue processing should be kept to a minimum, with sections prevented from dehydration and mounted in 85% glycerol. With proper preservation of myelin structure, BRM provides exquisitely detailed images that facilitate the assessment of myelin pathology associated with injury or disease.
