Development of a sample preparation protocol for 3D kidney ultrastructural analysis and immunolabeling by light microscopy

摘要 NIDDK肾脏精准医学项目（KPMP）等队列研究的成功 其大量的样本取决于肾脏的成本和时间效率评估 多尺度的活组织检查。在纳米级评估肾脏组织的结构（~10 nm），并在该背景下评估蛋白质生物标志物的3-D分布，然而， 目前严重限制，因为它依赖于昂贵，耗时和极其 复杂的相关光学和电子显微镜（CLEM）作为唯一合适的工具。泛光 开发了一种专有的新型组织扩张技术，我们称之为泛扩张 显微镜（pan-ExM），直接解决了这一挑战。通过结合约25倍线性 用氨基酸的新的本体（pan）染色扩增生物样品， 能够绘制特定生物标志物的三维纳米级分布， 肾脏超微结构标志性的超微结构特征，如足细胞足突， 线粒体中的嵴现在可以通过其特征性的泛染色模式来分辨， 类似于电子显微镜（EM）中的重金属染色。与EM/CLEM相比， 然而，pan-ExM是一种全光学方法，允许研究人员询问组织样本， 使用标准的共焦显微镜;这在时间，成本， 复杂性和样本保存与EM替代品的比较。我们建议的第一阶段工程， 开发并验证肾脏样本的泛ExM。具体而言，我们将：（1）优化和验证 在新鲜固定的肾组织中的新的抗体标记pan-ExM方案;以及（2）开发和 验证存档FFPE肾活检的新型~ 25 x组织扩张方案。这项工作是 旨在为第二阶段项目奠定基础，该项目将使泛ExM成为一个强大的， 强大的成像技术在肾脏领域，使每一个研究人员在肾脏， 将多种特异性蛋白质生物标志物定位在其3D背景区室中的约10 nm尺度。我们的长期目标是使泛ExM成为核心成像技术， 肾组织图谱以及用于靶向研究和诊断的优选技术 应用，从而为肾脏病的精准医学铺平道路。

Abstract The success of cohort studies such as the NIDDK Kidney Precision Medicine Project (KPMP) with its massive amount of samples depends on the cost and time-efficient evaluation of kidney biopsies at multiple scales. Assessing the structure of kidney tissue at the nanoscale (~10 nm) and evaluating the 3-D distribution of protein biomarkers in that context is, however, currently severely limited as it depends on expensive, time-consuming and extremely complex correlative light and electron microscopy (CLEM) as the only suitable tool. Panluminate has developed a proprietary new tissue expansion technology we call pan-Expansion Microscopy (pan-ExM), that directly addresses this challenge. By combining ~25-fold linear expansion of biological samples with novel bulk (pan) staining of amino acids, pan-ExM is capable of mapping the 3-D nanoscale distribution of specific biomarkers in the context of kidney ultrastructure. Hallmark ultrastructural features such as podocyte foot processes and cristae in mitochondria can now be resolved by their characteristic pan-staining pattern, analogous to heavy-metal stains in electron microscopy (EM). In contrast to EM/CLEM, however, pan-ExM is an all-optical method that allows researchers to interrogate tissue samples using standard confocal microscopes; this offers important advantages in terms of time, cost, complexity and sample preservation versus EM alternatives. Our proposed Phase I project is to develop and validate pan-ExM for kidney samples. Specifically we will: (1) optimize and validate a new antibody-labeling pan-ExM protocol in freshly fixed kidney tissue; and (2) develop and validate a novel ~25x tissue expansion protocol for archived FFPE kidney biopsies. This work is designed to lay the foundation for a Phase II project that will establish pan-ExM as a robust and powerful imaging technology in the kidney field by enabling every researcher in nephrology to locate multiple specific protein biomarkers within their 3D contextual compartments at the ~10 nm scale. Our long-term ambition is for pan-ExM to become a core imaging technology for kidney tissue atlases as well as a preferred technique for targeted research and diagnostic applications, thereby paving the road to precision medicine for nephrology.