1. Imaging early pathogenesis in glaucoma

Glaucoma is one of the leading causes of blindness worldwide, where the primary neurons in the retina, i.e. the retinal ganglion cells (RGCs), are gradually lost leading to blindness. While early detection of the disease is crucial to prevent loss of vision, it is hampered by the limitations of current techniques which cannot diagnose the disease until a significant portion of RGC axons (which make up the retinal nerves) have been irreversibly damaged. The pathogenic mechanisms are still poorly understood, but growing evidence suggests that microtubules (MTs) in the RGC axons are compromised during glaucoma progression. MT disruption may represent an early event in glaucoma pathogenesis, which could allow the disease to be detected much earlier than currently possible.

Our hypothesis is that MT disruption occurs before the loss of RGC soma or axon and that it is physiologically reversible. In order to confirm this hypothesis and unravel the mechanistic role of MTs in glaucoma, we developed second-harmonic generation (SHG), which arises specifically from MTs in the retinal nerves. It was for the first time that SHG was demonstrated as an intrinsic signal for retinal imaging. SHG can advance our knowledge of MT in axonal transport and cell motility, which so far relied largely on assays of MT reconstituted in vitro or in cultured cells. SHG allows MT interactions to be measured inside the cell in a condition close to native living tissue. Furthermore, the properties of SHG may allow new discoveries. Since the physical origin of SHG is distinguished from the previous contrasts, such as reflectance and birefringence, distinct information about the structure of MT can be attained from the SHG radiation. With this achievement, we are investigating the cytoskeleton in live tissue without exogenous labeling.

2. Imaging in living brain 1: Cortical myelination

Glia, far outnumbering neuron in the brain, is increasingly recognized for the complex roles beyond supporting the neural network. One of the important roles of glial cells in the vertebrate nervous systems is to produce and maintain the myelin sheath, a special multiple-layered membrane surrounding the axon. Proper conduction of neuronal impulses along the myelinated nerves hinges on the structure of myelin. Consequently, its formation and maintenance are tightly regulated through interactions between axon and myelin-forming glial cells, and abnormality could lead to clinical conditions including multiple sclerosis (MS) and Charcot-Marie-Tooth disease. However, the dynamics of axon-glial cell interaction underlying myelination and demyelination is poorly understood. It is difficult to study because of the current lack of technology to observe the myelin structure in vivo.

To address the bottleneck, we developed third-harmonic generation (THG) as an intrinsic nonlinear optical signal stemming from the myelin lamellae. We demonstrated a THG-based morphometry for precise estimation of g-ratio, a major biometric predictor of myelin function. The techniques allows us to ask important questions in the neurobiology of myelin, including the significance of cortical myelination in the development and diseases and the functional roles of prominent myelin domains, in particular of Schimidt-Lanterman incisure and Cajal band. We are now able to unravel, by means of THG, the organizing principle of myelin architecture and how it is violated to cause neurodegenerative disorders.

3. Imaging in living brain 2: Cell-type-specific transcription

Gene expression is a fundamental problem in biology. While all cells in an adult organism contain an identical set of genes, their expression varies considerably across cell types as well as upon different environmental cues, thus intricately controlled in space and time by hierarchies of complex regulation. The heterogeneity is thought to play significant roles in the development and function in the central nervous system (CNS), which consists of diverse cell types within a highly ordered architecture. In particular, noisy transcription in cortical neurons is likely to play significant roles not only in the normal functions of the brain but also in the pathogenesis of neurodegenerative disorders. However, understanding the mechanism has been hard due to the lack of methods to image the dynamics in the native environment.

To tackle this problem, we are developing new approaches involving molecular biology and multiphoton microscopy. In collaboration with Dr. Robert H. Singer, we demonstrated the visualization of dynamic gene expression in live mouse brain. We studied the synthesis of mRNAs in and their subsequent transport out of the nucleus, revealing previously unknown biophysics of mRNAs. Currently, we are interested in elucidating how in vivo transcription in the CNS occurs in a cell-type and cortical-layer specific manner.