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

总结 用于询问组织标本的临床金标准方法，基于载玻片（2D）的组织病理学，是基于 几个世纪前的技术有很多固有的局限性。最近的技术进步表明， 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 与传统的基于切片的组织学相比，非破坏性3D组织学具有提供 诊断病理学性能的变革性改进，原因如下：（1） （> 100 X）组织样本的采样，（2）3D细胞分布和组织结构的体积成像， 是预后和预测，（3）无损成像，这使得有价值的活检标本，以使用 用于下游生物标志物评估，以及（4）简化的过程，为医疗机构带来成本效益 和付款人。近年来，我们开发了一种技术，开放式光片（OTLS）显微镜， 能够对离体标本进行高通量无损3D组织学分析。我们的第一代OTLS 显微镜和成像协议证明了可靠地成像各种光学透明的临床 组织标本（手术切除和活组织检查）以无损方式进行，不会干扰 常规病理学方法。在这里，我们建议开发一种多分辨率混合OTLS显微镜（Aim 1），基于一种新型的非正交双目标（NODO）架构，这将是上级在各个方面 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/标记协议和样品架材料（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，以实现组织标记的自动化和标准化 以及用于H&E染色的稳健荧光类似物的透明化过程（目的2）。最后，我们将开发后- 用于厚组织的图像引导宏观解剖的成像技术，我们将展示其具有以下能力： 显著提高基因组测定的灵敏度（目的3）。总的来说，我们的项目旨在 将当前的2D病理学工作流程扩展到3D，以最大限度地减少临床采用障碍。快速翻译 通过3D病理学服务公司（Lightspeed Microscopy Inc.）授权我们 整个3D病理学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， 沿着用于临床决策支持（即，统计和预测）的AI分析方法的发展 治疗反应）。在这个项目中开发的仪器平台将直接支持一些 未来以疾病为重点的临床研究，以证明3D病理学在精确治疗 多种病症，如肾病、神经变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.