Instrumentation platform for 3D pathology with open-top light-sheet microscopy

摘要 临床询问组织标本的金标准方法，即基于载片的(2D)组织病理学，是基于 关于具有许多内在局限性的数百年的技术。最近的技术进步证明了 实现活检和手术标本的高通量无载玻片3D组织学的可行性。在……里面 与传统的基于玻片的组织学相比，非破坏性3D组织学具有提供 诊断病理学表现的变革性改进有以下几个原因：(1)大大提高 (&gt；100X)组织样本采样，(2)3D细胞分布和组织结构的体积成像， 预后和预测性；(3)非破坏性成像，允许使用有价值的活检标本 用于下游生物标记物评估，以及(4)为医疗机构提供具有成本效益的简化流程 和付款人。近年来，我们开发了一种技术，开顶式光片显微镜(OTLS)，以 实现体外标本的高通量非破坏性3D组织学。我们的第一代OTLS 显微镜和成像方案证明了能够可靠地成像各种光学清晰的临床 组织标本(手术切除和活组织检查)以非破坏性的方式进行，不干扰 常规病理学方法。在这里，我们提出了一种多分辨率混合OTLS显微镜(AIM 1)，基于一种新的非正交双目标(NoDo)架构，该架构在各方面都将是优越的 对于我们以前的系统，包括分辨率(和分辨率范围)、成像深度以及与 几乎所有的清除/标记方案和样品保持器材料(对折射率不匹配不敏感)。 此外，我们将开发创新的预成像方法，使组织标记自动化和标准化 和H&E染色的强健荧光类似物的清除过程(目标2)。最后，我们将发展后 用于图像引导的厚组织宏观解剖的成像技术，我们将展示这些技术具有 显著提高基因组分析的灵敏度(目标3)。总体而言，我们的项目目标旨在 将当前的2D病理工作流程扩展到3D，以最大限度地减少临床采用障碍。一种快速的翻译 PATH通过一家3D病理服务公司存在(光速显微镜公司)它已经授权我们的 完整的3D病理IP组合。作为这一更大的转化努力的一部分，我们的实验室正在进行临床研究， 随着用于临床决策支持(即预测和预测)的人工智能分析方法的发展 治疗反应)。本项目开发的仪器平台将直接支持多个 未来以疾病为重点的临床研究，以证明3D病理学在精准治疗食道癌中的价值 各种疾病，如肾脏疾病、神经退行性疾病和各种形式的癌症。

Summary The clinical gold-standard method for interrogating tissue specimens, slide-based (2D) histopathology, is based on centuries-old technologies with many inherent limitations. Recent technological advances have demonstrated the feasibility of achieving high-throughput slide-free 3D histology of biopsy and surgical specimens. In comparison to conventional slide-based histology, nondestructive 3D histology has the potential to provide a transformative improvement in diagnostic pathology performance for a number of reasons: (1) vastly greater (>100X) sampling of tissue specimens, (2) volumetric imaging of 3D cell distributions and tissue structures that are prognostic and predictive, (3) nondestructive imaging, which allows valuable biopsy specimens to be used for downstream biomarker assessment, and (4) a simplified process with cost benefits for healthcare institutions and payers. In recent years, we have developed a technology, open-top light-sheet (OTLS) microscopy, to enable high-throughput nondestructive 3D histology of ex vivo specimens. Our first generations of OTLS microscopes and imaging protocols demonstrated the ability to reliably image a variety of optically cleared clinical tissue specimens (surgical excisions and biopsies) in a nondestructive manner that does not interfere with conventional pathology methods. Here, we propose to develop a multi-resolution hybrid OTLS microscope (Aim 1), based on a novel non-orthogonal dual-objective (NODO) architecture, which will be superior in every regard to our previous systems, including resolution (and range of resolutions), imaging depth, and compatibility with nearly all clearing/labeling protocols and sample-holder materials (insensitivity to refractive-index mismatch). Furthermore, we will develop innovative pre-imaging methods to automate and standardize the tissue-labeling and clearing process for a robust fluorescent analog of H&E staining (Aim 2). Finally, we will develop post- imaging technologies for image-guided macro-dissection of thick tissues, which we will show has the ability to significantly improve the sensitivity of genomic assays (Aim 3). Collectively, our project aims are designed to extend current 2D pathology workflows into 3D to minimize clinical-adoption barriers. A rapid translational pathway exists through a 3D-pathology-services company (Lightspeed Microscopy Inc.) that has licensed our entire 3D pathology IP portfolio. As part of this larger translational effort, clinical studies are ongoing in our labs, along with development of AI-analysis methods for clinical decision-support (i.e. prognostication and prediction of treatment response). The instrumentation platform developed in this project will directly support a number of future disease-focused clinical studies to demonstrate the value of 3D pathology for the precision treatment of diverse conditions such as kidney disease, neurodegenerative diseases, and various forms of cancer.