The mission of Lim Lab is to tackle outstanding problems in life science using novel imaging technology. We invent new tools ‘on demand’; instead of developing generic methods, first we identify the most important question and then address the current bottleneck hampering our understanding by enabling previously impossible measurements.
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 eventually 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, I developed a novel imaging contrast of second-harmonic generation (SHG). We showed that SHG arises specifically from MTs in the retinal nerves and it can be employed for visualization and quantification of the morphology. Although widely used for biological imaging, it was for the first time that SHG was demonstrated for retinal imaging. With microtubule SHG, we are now equipped with a new imaging contrast that allows the cytoskeleton to be investigated in live tissue. Our present knowledge of MT in axonal transport and cell motility is largely from 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 offer unique opportunities that have never been explored. The physical origin of SHG is distinguished from the previous contrasts, such as reflectance and birefringence, on which the current retinal imaging modalities are based. As a result, distinct information about the structure of MT can be attained from the SHG radiation.
H. Lim and J. Danias, “Label-free morphometry of retinal nerve fiber bundles by second-harmonic generation microscopy”, Opt. Lett. 37, 2316-2318 (2012).
H. Lim and J. Danias, “Effect of axonal microtubules on the morphology of retinal nerve fibers studied by second-harmonic generation”, J. Biomed. Opt. 17, 110502 (2012).
H. Lim, M. Mujat, C. Kerbage, E. C. Lee, Y. Chen, T. C. Chen, and J. F. de Boer, " High-speed imaging of human retina in vivo with swept-source optical coherence tomography," Opt. Express 14, 12902-12908 (2006).
2. Imaging transcription in living animals
Gene expression is a problem at the core of cellular biology. All cells in an adult organism contain an identical set of genes but their expression varies depending on the cell type and environment. The process is intricately controlled in space and time by hierarchies of complex regulation. Here we are interested in the biophysical basis of the variabilities in gene expression in the central nervous system (CNS), which is characterized by the diversity of constituent cell types as well as highly organized tissue architectures such as cortical columns. Evidence suggests that transcription in cortical neurons is likely to play significant roles not only in the functional modulation of neuronal network but also in the pathogenesis of neurodegenerative disorders. However, the mechanistic understanding has been hard to acquire due to the lack of suitable methods to image the dynamic process as it occurs in the native environment. To address this bottleneck, we are developing new approaches involving molecular biology, protein engineering, and multiphoton microscopy. In collaboration with Dr. Robert Singer at Albert Einstein College of Medicine, we recently demonstrated the visualization of dynamic gene expression in live mouse brain tissue. In our recent study we interrogated the dynamics of the single molecules, e.g. transcription and transport of mRNA granules, revealing previously unknown biophysics of mRNA. Our current efforts are focused on developing more versatile methodologies to ask the fundamental question in molecular cell biology, e.g. how in vivo transcription in the CNS occurs in a cell-type and cortical-layer specific manner.
H.Y. Park, H. Lim, Y,J. Yoon, A. Follenzi, C. Nwokafor, M. Lopez-Jones, X. Meng, and R.H. Singer, “Visualization of Dynamics of Single Endogenous mRNA as Labeled in Live Mouse”, Science 343, 422-424 (2014).
3. Imaging myelination in vivo
Glia, far outnumbering neuron in the brain, is increasingly recognized for the roles more than 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 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 issue, we developed a technique based on an intrinsic nonlinear optical signal stemming from the myelin lamellae, namely third-harmonic generation (THG). We demonstrated a THG-based morphometry for precise estimation of g-ratio, a major biometric predictor of myelin function. The developed techniques places us at a vantage point 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 imaging, the organizing principle of myelin architecture and how it is violated to cause neurodegenerative disorders.
H. Lim, D. Sharoukhov, I. Kassim, Y. Zhang, J.L. Salzer, C. V. Melendez-Vasquez, "Label-free imaging of Schwann cell myelination by third harmonic generation microscopy", Proc. Natl. Acad. Sci. U.S.A. 111, 18025-18030 (2014).
Supported by NIH and the BrightFocus Foundation