Title: Development of advanced Raman microscopy methods to interrogate the brain
Description: A central quest in biology is to understand the structure-function relationship of complex biological systems. The brain represents the ultimate complexity of a biological system: (1) the vertebrate brain contains 107-1011 neurons interconnected with glial cells; (2) over tens of diverse cell types are organized in a hierarchical way over an intricate landscape; (3) coordinated electrical and chemical activities of neuronal ensembles generate emergent properties and functions; and (4) each neuron can extend over large volumes with its spatial scales spanning 6 orders of magnitude. As a result, compared to other organ systems, our understanding of the brain remains primitive and obscure in terms of both its structures and its functions. Accordingly, many grand challenges endure in brain sciences, including comprehensively mapping neuronal wiring of the brain, an exhaustive taxonomy of cell types in the brain, and robust diagnostic and therapeutic strategies for brain diseases. These challenges are difficult to tackle with existing microscopy methods, because general trade-offs prevail between number of colors, imaging depth, spatial resolution, imaging throughput, sensitivity, and specificity.

Therefore, the quest to understand the brain calls for advances and innovations on novel microscopy methods.The evolution of modern Raman microscopy is fundamentally driven by the development of novel spectroscopy methods. The advancement of molecular spectroscopy in turn pushes forward and benefits from, the progress in vibrational probes, labeling chemistry, and sample processing and transformation. In particular, stimulated Raman scattering (SRS) microscopy offers high sensitivity and fast acquisition for biomedical imaging, by harnessing accelerated vibrational transition from stimulated emission. Bio-orthogonal chemical imaging provides chemical specificity and minimal perturbation for visualizing metabolic dynamics of small molecules, by using tiny vibrational probes such as deuterium and alkyne. Electronic pre-resonance SRS (epr-SRS) microscopy further enhances the sensitivity to the nanomolar level for imaging specific proteins, by exploiting electronic pre-resonance of specially designed Raman dyes.

Despite these notable innovations, the imaging depth of these Raman microscopy methods is limited to superficial layers of biological tissues (~100 μm) due to light scattering. This dissertation contributes to the development of advanced Raman microscopy methods for volumetric imaging with extended imaging depth in scattering tissues. For this purpose, we develop a set of tissue clearing strategies tailored to specific Raman imaging modalities. In addition, we develop image analysis methods to extract systems information from volumetric high-dimensional imaging datasets. Equipped with our volumetric imaging and analysis methods, we elucidate intricate structures and functions of the brain at both physiological and pathological conditions, providing implications for brain tumor metabolism and cerebellum development. Chapter 1 introduces an overview of Raman microscopy with particular emphasis on SRS and epr-SRS microscopies. Chapter 2 discusses the principles of tissue clearing with special focus on the basis of light scattering, the working mechanisms of different categories of tissue clearing methods, and the rationale underlying the development and evolution of these tissue clearing methods.

Chapter 3 describes the development of volumetric chemical imaging, which brings label-free SRS microscopy, bio-orthogonal chemical imaging, and metabolic imaging to the realm of volumetric imaging with greater than 10-fold depth extension. Chapter 4 depicts the development of volumetric multiplex imaging, which generalizes epr-SRS microscopy to the territory of volumetric imaging. With this method we achieve one-shot imaging of more than 10 colors over millimeter-thick brain tissues, extending the imaging depth of multiplex protein imaging by 10~100 folds. Chapter 5 is a manuscript of an ongoing project on imaging nanocarriers for drug delivery across the blood-brain barrier (BBB). We develop a method of correlative multispectral SRS and fluorescence microscopy to image nanoparticles by SRS with multispectral information and particle counting capability and to image tissue context (especially cerebral vasculature) by fluorescence with high specificity. Using this method, we achieve direct imaging of nanocarriers that cross the BBB with definitive spectral evidence and single particle sensitivity.

The preliminary results quantifying the proportion of nanoparticles that cross the BBB provide implications that challenge the current understanding of drug delivery to the brain.