Coord Core - Atkinson

淋巴系统有五个主要功能：1）清除多余的体液; 2）吸收和运输 脂肪酸/乳糜的循环系统; 3）血液过滤; 4）安装主要防御 感染和癌症;以及5）免疫细胞的产生和激活， 调节性免疫细胞保护自身免疫/自身炎症疾病。鉴于这些关键 生理功能，我们认为重要的是开发三个主要的三维（3D）组织图 人类免疫系统的淋巴器官：脾、胸腺和淋巴结（器官特异性项目1- 3、分别）。作为我们的主要优势之一，我们拥有超过10年的采购经验， 移植质量的器官用于研究，包括淋巴系统。作为这一24/7/365努力的一部分， 我们与美国器官采购组织（OPO）建立了良好的关系， 56/58在过去的十年里。我们的标准操作程序（SOP），包括发布的和基于网络的， 包括正常性评估、质量控制测定和系统解剖解剖/储存。我们 还参与国家质量保证/质量控制计划，以评估组织处理/库存程序。我们的方法 为了朝着3D组织图的共同目标开发测定管道，最初将涉及获取 完整组织的宏观图像，并利用我们在磁共振方面的优势， 成像（MRI）。拟议的光学显微镜管道将使用福尔马林解决微观解剖特征 固定石蜡包埋（FFPE）和最佳切割温度（OCT）复合包埋切片 作为组织光学透明化和扩张。这些标本将研究从纳米到毫米的分辨率使用 随机光学重建显微镜（STORM），共焦，多光子和光片荧光 显微镜（LSFM），所有管道共享一个通用的文件格式，以简化3D重建。基于 关于淋巴器官在免疫细胞产生和运输中的独特作用，荧光激活 来自血液和每个淋巴器官的细胞的细胞分选（FACS）将提供患者的比较- 特异性免疫细胞库，并用于随后的单细胞RNA-seq分析。共同注册 为了将生物分子与其同源细胞结合，我们将采用成像质谱细胞术（IMC），多重单分子 荧光原位杂交（smFISH）和多重错误鲁棒FISH（MERFISH），以映射细胞 蛋白质和mRNA表达，最终在每个3D组织图谱上。我们在器官采购方面的经验， 国家高磁场实验室（NHML）高级磁共振成像的可用性 和光谱学（AMRIS）设施在UF，多光子/共聚焦显微镜，LSFM，最先进的流式细胞仪，10倍 Genomics Chromium控制器和大学RNA-Seq的高通量cDNA文库测序 佛罗里达大学的IMC和苏黎世大学的3D数据分析设施， 使我们能够提供最高质量的淋巴器官3D人体生物分子图谱（HuBMAP）。

The lymphatic system serves five primary roles: 1) removal of excess body fluids; 2) absorption and transport of fatty acids/chyle to the circulatory system; 3) blood filtration; 4) mounting the primary defense against infections and cancer through immune cell production and activation; and 5) generation and activation of regulatory immune cells that protect against autoimmune/autoinflammatory disease. Given these key physiological functions, we deem it important to develop a three-dimensional (3D) tissue map for three major lymphoid organs of the human immune system: spleen, thymus and lymph nodes (Organ Specific Projects 1- 3, respectively). As one of our primary strengths, we have over 10 years of experience in the procurement of transplant-quality organs for research, including those of the lymphatic system. As part of this 24/7/365 effort, we have well-established relationships with the U.S. Organ Procurement Organizations (OPO), having referrals from 56/58 over the last decade. Our standard operating procedures (SOPs), both published and web-based, include assessment of normality, quality control assays, and systematic anatomical dissection/storage. We also participate in National QA/QC programs to evaluate tissue processing/banking procedures. Our approach to developing assay pipelines towards the common goal of a 3D tissue map will initially involve acquiring a macro image of the intact tissue and addressing tissue morphology, using our strengths in magnetic resonance imaging (MRI). The proposed optical microscopy pipeline will address microanatomical features using formalin fixed paraffin embedded (FFPE) and optimal cutting temperature (OCT) compound embedded sections as well as tissue optical clearing and expansion. These specimens will be studied from nm to mm resolution using stochastic optical reconstruction microscopy (STORM), confocal, multiphoton and light sheet fluorescence microscopies (LSFM), with all pipelines sharing a common file format for simplified 3D reconstruction. Based on the unique role for lymphatic organs in production and trafficking of immune cells, fluorescence activated cell sorting (FACS) of cells from blood and each lymphatic organ will provide a comparison of the patient- specific immune cell repertoire and serve in subsequent single cell RNA-seq analyses. To co-register biomolecules to their cognate cells, we will employ imaging mass cytometry (IMC), multiplex single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) to map cellular protein and mRNA expression, ultimately on each 3D tissue atlas. Our experience in organ procurement, the availability of the National High Magnetic Field Laboratory (NHML) Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility at UF, multiphoton/confocal microscopy, LSFM, state of the art FACS, 10X GENOMICS Chromium controller, and high throughput cDNA library sequencing for RNA-Seq at the University of Florida, as well as world leading IMC and 3D data analysis facility at the University of Zurich, ideally positions us to provide the highest quality 3D Human BioMolecular Atlas (HuBMAP) of the lymphatic organs.