Correlative nonvital standard histopathology and vital multiphoton prehistopathology

总结 医学上处理新鲜活检的人体组织的一种常见做法是将其迅速放入福尔马林中 溶液这种“固定”保护组织免于腐烂，允许运输到（远程）病理 用于标准组织病理学或其他生化/遗传分析的实验室。但是福尔马林 “杀死”组织，因此与其生命状态相关的生化和代谢功能被永久性地破坏。 迷路了这些功能可能包含标准组织病理学无法提供的诊断信息，例如 在已知的结构和化学扰动之前， 常规组织学样本处理。因此，对“组织活力”进行非侵入性成像将是非常有益的 并且在固定之前快速地（在几分钟内）通过新颖的光学成像技术。而其中也有很多 为此目的，已经开发了一些技术，它们的诊断能力独立于标准 组织病理学尚未明确证明，由于缺乏相关技术， 配准/共同定位活体组织的虚拟光学切片（光学成像）和实际的福尔马林固定的光学切片。 经处理/非活体组织的石蜡包埋切片（标准组织病理学）。因此，通常很难 以证明在临床标准护理过程中新型光学成像技术的额外成本是合理的。 在这个项目中，我们的目标是发展标准组织病理学（在一个 线性/单光子光学机制）和基于多光子过程的新型光学成像技术， 即多光子预组织病理学。后者已在我们先前的工作中得到证明， 未标记的内源性生物分子和片段丰富的细胞和细胞外组分。的 所提出的相关显微镜将追溯使用该方法成像的新鲜离体组织的光学切片。 无标记多光子预组织病理学检查，以相应的福尔马林固定石蜡包埋切片， 不同的组织学染色在概念验证示范（第一阶段）中，我们将使用丢弃的新鲜啮齿动物 组织标本，以模拟新鲜活检的人体组织标本，并配准大面积（1x 1 mm 2）的 多光子预组织病理学成像与标准苏木精和伊红（H&E）成像 组织学在未来阶段（第二阶段），我们将把配准面积从1x 1 mm 2扩大到10 x10 mm 2， 适应更大的样本量，将染色从H&E扩展到各种免疫组织学染色， 在临床环境中，将肉类产品的样本转换为新鲜的人体组织活检。成功 该项目的成果将为病理学家提供一个实时、无标签、无载玻片、数字化和即时的 或程序点工具来成像“组织活力”，而不影响现有的组织学工作流程， 新增加的关键信息，以补充基于非重要组织的金标准。

SUMMARY A common practice in medicine for handling freshly biopsied human tissue is to rapidly put it into a formalin solution. This “fixation” preserves the tissue from decay, allowing transportation to a (remote) pathology laboratory for standard histopathology or other biochemical/genetical analyses. However, formalin instantly “kills” the tissue, so that the biochemical and metabolic functions associated with its vital state are permanently lost. These functions likely contain diagnostic information not available from standard histopathology, e.g. the pristine picture of an active tumor microenvironment before the structural and chemical perturbations known in routine histological sample treatments. Thus, it will be highly beneficial to image “tissue vitality” noninvasively and rapidly (within minutes) before the fixation, by a novel optical imaging technology. Although numerous technologies have been developed for this purpose, their diagnostic capabilities independent from standard histopathology have not been unambiguously demonstrated, due to the lack of a correlative technology to co- register/co-localize the virtual optical sections of vital tissue (optical imaging) and the actual formalin-fixed paraffin-embedded sections of treated/nonvital tissue (standard histopathology). As a result, it is often difficult to justify the additional cost of novel optical imaging technologies in clinical standard-of-care processes. In this project, we aim to develop correlative microscopy between standard histopathology (operated in a linear/single-photon optical regime) and a novel optical imaging technology based on multiphoton processes, i.e. multiphoton pre-histopathology. The latter has been demonstrated in our prior work to visualize multiple unlabeled endogenous biomolecules and segment a rich set of cellular and extracellular components. The proposed correlative microscopy will retrace optical sections of fresh ex vivo tissue that were imaged using the label-free multiphoton pre-histopathology to the corresponding formalin-fixed paraffin-embedded sections with diverse histological stains. For proof-of-concept demonstration (Phase I), we will use discarded fresh rodent tissue specimens to mimic freshly biopsied human tissue specimens, and co-register a wide area (1x1 mm2) of image in the multiphoton pre-histopathology with its counterpart in standard hematoxylin and eosin (H&E) histology. In a future stage (Phase II), we will expand the co-registration area from 1x1 mm2 to 10x10 mm2 to accommodate larger sample sizes, extend the stains from H&E to diverse immunohistological stains, and switch the samples from the meat products to fresh human tissue biopsies in a clinical setting. The successful outcome of this project will equip pathologists with a real-time, label-free, slide-free, digital, and point-of-care or point-of-procedure tool to image “tissue vitality”, without affecting the existing histology workflow but with newly added critical information to complement the gold standard based on nonvital tissue.