Advanced Expansion Microscopy Imaging System for Brain Microglia

先进的脑小胶质细胞扩张显微镜成像系统

• 批准号: RTI-2021-00113
• 负责人: Ciernia, Annie
• 金额: $ 10.93万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718782
• 关键词: Advanced; Expansion; Microscopy; Imaging; System

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.

神经系统和免疫系统之间的相互作用对正常的大脑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，以控制神经元的数量和连接。小胶质细胞还形成成人大脑中学习和记忆所需的神经元连接。由于突触的小尺寸（<1 μ m 3）和标准共聚焦显微镜的低通量能力，对神经元突触的小胶质细胞相互作用和吞噬作用（修剪）的成像非常有限。我们最近已经克服了第一个限制，使用扩展显微镜（ExM），扩大组织~4倍，同时保留感兴趣的生物分子。通过将脑组织的ExM与小胶质细胞（P2YR12）、激活状态（CD68溶酶体激活）和来自不同神经元亚型（例如，多巴胺能、GABA能）的突触标记物的染色相结合，我们将能够鉴定和量化发育和成年期特定神经元回路的小胶质细胞修剪。 ExM是革命性的，因为它与高通量成像平台兼容，如蔡司细胞发现者7（CD7）。因此，我们正在申请资金，用于一种新型的、高通量的成像设置，并为ExM优化物镜。CD7比共焦成像快30倍，对于新手用户来说更容易。数百个小胶质细胞可以在数小时内成像，突触吞噬作用定量，而目前的速度为数周。这有助于跨脑区域、性别、年龄和治疗的小胶质细胞修剪的有效、高置信度、定量分析。目前，在我们的机构没有可行的替代方案用于小胶质细胞吞噬作用的ExM高通量分析。这种最先进的技术只能通过拟议的成像设置来充分利用，并将使UBC成为小胶质细胞生物学的领导者。 该设备对于三个NSERC相关研究项目至关重要，所有这些项目都旨在确定免疫细胞如何促进正常的大脑功能。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层内神经炎症对高阶认知的贡献。共同申请人拥有科学专业知识和运营成本支持，可以充分利用所提议的成像设置。Ciernia、Galea和Winstanley总共有39名受训人员将使用这些设备，其中一半受训人员直接从事与NSERC有关的工作。HQP将获得ExM组织制备，染色，图像采集，分析，统计和手稿准备的技能，所有这些对于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。