Advanced Expansion Microscopy Imaging System for Brain Microglia

神经系统和免疫系统之间的相互作用对大脑的正常发育和功能至关重要。小胶质细胞是大脑的免疫细胞，在大脑发育过程中直接与神经元相互作用，控制神经元的数量和连接。小胶质细胞还塑造成人大脑学习和记忆所需的神经元连接。由于突触的小尺寸(1um3)和标准共聚焦显微镜的低穿透能力，小胶质细胞的相互作用和神经元突触的吞噬(修剪)成像一直是极其有限的。最近，我们使用扩展显微镜(EXM)克服了第一个限制，它在保留感兴趣的生物分子的同时将组织扩展到原来的4倍。通过结合脑组织的EXM和小胶质细胞(P2YR12)、激活状态(CD68溶酶体激活)和不同神经元亚型的突触标记(如5-羟色胺能、GABA能)的染色，我们将能够识别和定量小胶质细胞在发育和成年期特定神经元回路的修剪。 Exm是革命性的，因为它与高通量成像平台兼容，例如蔡司细胞发现者7(CD7)。因此，我们正在为一种新颖的、高通量的成像设备申请资金，并优化了exm的目标。CD7的成像速度比共焦成像快30倍，对于新手来说要容易得多。与目前几周的速度相比，数百个小胶质细胞可以在几个小时内成像并量化突触吞噬作用。这有助于对跨脑区域、性别、年龄和治疗的小胶质细胞修剪进行高效、高置信度、定量的分析。目前，我们研究所还没有可行的替代方法来高通量分析小胶质细胞吞噬功能。这项最先进的技术只有通过建议的成像设备才能被充分利用，并将使UBC成为小胶质细胞生物学领域的领导者。 该设备对三个与NSERC相关的研究项目至关重要，所有这些项目都旨在确定免疫细胞如何对正常的大脑功能做出贡献。1)Ciernia的工作将确定大脑中的先天免疫细胞如何控制大脑发育；2)Galea的工作将确定雌二醇是否调节神经炎症以影响认知；3)Winstanley的工作将确定前额叶皮质内的神经炎症对高级认知的贡献。共同申请者拥有科学专业知识和运营成本支持，可以充分利用拟议的成像设备。Ciernia、Galea和Winstanley总共有39名学员可以使用这种设备，其中一半直接从事与NSERC相关的工作。HQP将获得EXM组织准备、染色、图像获取、分析、统计和手稿准备方面的技能，所有这些技能对学术界内外竞争激烈的职业生涯都很重要。NSERC资助的所有三个研究项目中的HQP将直接受益于这种分析小胶质细胞修剪的新方法。

Interactions between the nervous and immune systems are critical for normal brain development and function. Microglia, the brain's immune cells, directly interact with neurons during brain development to control neuronal numbers and connections. Microglia also shape neuronal connections required for learning and memory in the adult brain. Imaging microglial interactions and phagocytosis (pruning) of neuronal synapses has been extremely limited due to the small size of synapses (<1um3) and the low throughput abilities of standard confocal microscopy. We have recently overcome the first limitation using Expanded Microscopy (ExM) that expands the tissue ~4x while preserving biomolecules of interest. By combining ExM of brain tissues with staining for microglia (P2YR12), activation state (CD68 lysosomal activation) and markers of synapses from different neuronal subtypes (e.g.serotonergic, GABAergic), we will be able to identify and quantify microglial pruning of specific neuronal circuits in development and adulthood. ExM is revolutionary in that it is compatible with high throughput imaging platforms, such as the Zeiss Cell Discoverer 7 (CD7). Thus, we are requesting funds for a novel, high throughput imaging setup with optimized objectives for ExM. The CD7 is 30x faster than confocal imaging and significantly easier for novice users. Hundreds of microglia can be imaged and synaptic phagocytosis quantified within hours, compared to the current rate of weeks. This facilitates the efficient, high confidence, quantitative analysis of microglial pruning across brain regions, sexes, ages and treatments. Currently, there is no viable alternative at our institution for ExM high throughput analysis of microglial phagocytosis. This state-of-the-art technique can only be fully utilized by with the proposed imaging setup and will establish UBC as a leader in microglial biology. This equipment is vital for three NSERC-related research programs, all of which aim to establish how immune cells contribute to normal brain function. 1) Ciernia's work will establish how innate immune cells in the brain control brain development 2) Galea's work will establish whether estradiol modulates neuroinflammation to impact cognition, and 3) Winstanley's work will establish the contribution of neuroinflammation within the prefrontal cortex on higher-order cognition. The co-applicants have the scientific expertise and operating cost support to fully utilize the proposed imaging setup. Together Ciernia, Galea and Winstanley have 39 trainees that will have access to this equipment, with half of these trainees directly engaged in NSERC-related work. HQP will acquire skills in ExM tissue preparation, staining, image acquisition, analysis, statistics and manuscript preparation, all important for highly competitive careers in and outside of academia. HQP in all three NSERC funded research programs will directly benefit from this new approach to analyzing microglial pruning.