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 其庞大的样本量取决于肾脏的成本和时效性评估 多个级别的活组织检查。在纳米尺度(~10)评估肾组织的结构 NM)，并且在这种情况下评估蛋白质生物标记物的三维分布是， 目前受到严重限制，因为它依赖于昂贵、耗时和极端 复合相关光电子显微镜(CLEM)是唯一合适的工具。泛鲁米特 开发了一种专利的新组织扩张技术，我们称之为泛扩张 显微镜(PAN-EXM)，它直接解决了这一挑战。通过将~25倍的线性 用新的氨基酸整体(PAN)染色扩大生物样品，PAN-EXM是 能够绘制特定生物标记物的3-D纳米尺度分布 肾脏超微结构。标志性超微结构特征，如足细胞足突和 线粒体中的隆起现在可以通过它们特有的泛染色图案来分辨， 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 使用标准共焦显微镜；这在时间、成本、 复杂性和样本保存与EM替代方案的对比。我们建议的第一阶段项目是 开发和验证肾脏样本的PAN-EXM。具体来说，我们将：(1)优化和验证 一种新的抗体标记的PAN-EXM方案在新固定的肾组织中；以及(2)开发和 验证一种新的~25倍组织扩张方案，用于存档的FFPE肾活检。这项工作是 旨在为第二阶段项目奠定基础，该项目将使泛美进出口银行成为一个强大和 肾脏领域强大的成像技术，使每一位肾脏内科的研究人员 将多个特定的蛋白质生物标记物定位在其3D上下文隔间中 纳米比例尺。我们的长期抱负是让PAN-EXM成为 肾组织图谱以及用于靶向研究和诊断的首选技术 应用，从而为肾脏病的精确医学铺平了道路。

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.