Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.

Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.

<p dir="ltr">The <i>SCN2A</i><i> </i>gene encodes for the neuronal sodium channel Na_V1.2, which mediates action potential initiation and propagation (Sanders et al., 2018). This protein is expressed mainly in the proximal axonal initial segment (AIS) and soma of glutamatergic excitatory cortical neurons (Kruth, Grisolano, Ahern, & Williams, 2020). <i>SCN2A</i> pathogenic variants have been associated with epilepsy. An example is the recurrent Nav1.2-L1342P variant, a heterozygous missense variant (Begemann et al., 2019) identified in five patients worldwide presenting an early-onset severe seizure phenotype that remains hard to treat with current medications (Que et al., 2021). Additionally, it is one of the few rare <i>SCN2A</i> variants that can impact brain structure (Miao et al., 2020).</p><p dir="ltr">Given that no disease-modifying treatment exists, there is an urgent need to generate novel tools to probe at variant-specific disease mechanisms, evaluate therapeutic interventions, and study interactions with other cell types. Previously, we demonstrated that hiPSC-derived 2D neuronal monolayers carrying the CRISPR/Cas9-edited Nav1.2-L1342P variant display a distinct hyperexcitability phenotype (Que et al., 2021). Despite these findings, questions persist regarding the Nav1.2-L1342P variant's influence on neurodevelopment in more physiologically relevant 3D models, such as organoids.</p><p dir="ltr">To address this, in Chapter 2 of this study, we generated human-induced pluripotent stem cell-derived cortical organoids carrying the epilepsy-related Nav1.2-L1342P variant to study its effect on neuronal hyperexcitability, neurodevelopment and other disease phenotypes. Our data suggests that Nav1.2-L1342P cortical organoid neurons display<b> </b>enhanced repetitive action potential firings, intrinsic excitability, enhanced calcium signaling, increased network neuronal firing, and excitatory postsynaptic currents (EPSCs), suggesting a marked hyperexcitability phenotype and enhanced excitatory neurotransmission. Moreover, cortical organoids with the Nav1.2-L1342P variant display significant changes in synaptic, glutamatergic, and development-related pathways. We also observed that Nav1.2-L1342P variant impacts cortical organoid synaptic and neuronal content.</p><p dir="ltr">The impact of the Nav1.2-L1342P variant was also demonstrated in the 2D-cortical neuron monolayer model, presenting a noticeable reduction in neuronal complexity, thus offering intriguing insights into their effect on neuronal morphology and developmental processes. Our findings recapitulate the hyperexcitable phenotype trends previously observed in the 2D-cortical neuron monolayer platform (Que et al., 2021) and provide evidence of non-autonomous cell development changes due to the Nav1.2-L1342P variant.</p><p dir="ltr">Chapter 3 of this dissertation established a co-culture of hiPSC-derived neurons and microglia, the brain's resident immune cells. Microglia originate from a different lineage (yolk sac) and are not naturally present in hiPSC-derived neuronal cultures. Therefore, they must be added to neuronal cultures to yield a heterogeneous environment. Microglia are also one of the few cell types able to respond to neuronal hypo and hyperexcitability changes. This unique capability prompted us to study how microglia responded to human neurons carrying a disease-causing variant and influenced neuronal excitability.</p><p dir="ltr">We found that microglia display increased branch length and enhanced process-specific calcium signal when co-cultured with the Nav1.2-L1342P neurons, recapitulating phenomena previously observed in rodent seizure models (Eyo et al., 2014; Nebeling et al., 2023). Moreover, the presence of microglia significantly lowered the repetitive action potential firing and current density of sodium channels in neurons carrying the variant, demonstrating the microglial capacity to influence and ameliorate the neuronal activity of the Nav1.2-L1342P mutant neurons. We hypothesized that this effect could be attributed to the increased release of glutamate or small molecules by the Nav1.2-L1342P mutant neurons, which could likely be triggering microglial responses. Additionally, we showed that co-culturing with microglia reduced sodium channel expression within the axon initial segment (AIS) of Nav1.2-L1342P neurons, explaining, in part, the mechanism behind the reduction of sodium current density.</p><p dir="ltr">Taken together, our observations with 2D cortical neurons and 3D cortical organoids revealed marked hyperexcitability and developmental changes associated with the Nav1.2-L1342P variant. Our work also reveals the critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing SCN2A variant.</p>