Label-free, high resolution, functional, two-photon imaging for non-invasive early cancer detection

项目总结 已建立的早期癌症检测方法依赖于简单的组织可视化方法，伴随着 活检和组织病理学评估，主要基于组织形态特征。这些 方法是不准确或低效的。我们的长期目标是将上皮癌前期和早期转化为 通过使用功能性代谢、形态和生物力学组织生物标记物进行诊断 从基于光纤探头的内源双光子中无创、自动和近实时地提取 图像。内源性双光子成像是唯一能够提供无标记、功能性、高分辨率的成像技术 组织图像。在这项提案中，我们的目标是建立和验证这些测量和生物标记物 用新鲜切除的组织检测人类宫颈癌前病变。宫颈是发育的理想器官。 并测试我们的方法，因为它放宽了内窥镜应用程序的一些大小限制， 使我们能够集中展示这一创新平台的原则。此外，我们预计我们的 所提出的方法将使宫颈癌前病变检测的特异性显著提高。至 为了达到我们的目标，我们将从50个新鲜切除的人类宫颈组织样本中获取图像 接受阴道镜检查、电环切除术或子宫切除术的患者。我们将获得 内源双光子激发荧光(TPEF)和二次谐波(SHG)图像和 提取与NADH、FAD和胶原蛋白有关的信号。我们将使用我们使用的方法来处理这些图像 开发用于评估：a)依赖深度的光学氧化还原比、线粒体组织和核比 上皮FAD和NADH TPEF图像的细胞质比率变化，以及b)胶原纤维 基质的SHG和TPEF图像的组织和交联。我们将使用判别分析来 开发算法，包括提取的光学参数的最佳组合，以区分高 从宫颈病变分级非高级别，以组织病理学为金标准。我们的算法将 完全自动化和快速，并将产生基于功能组织特征的诊断。因此，我们 预计这项研究的结果将推动基于探针的2P成像系统的临床开发 体内成像翻译，使实时、高精度检测宫颈癌前病变成为可能。 最终，我们预计基于探针的2P成像将改变广泛范围内的早期癌症诊断 组织，如口腔、食道、结肠和膀胱。

PROJECT SUMMARY Established methods for early cancer detection rely on simple tissue visualization methods, accompanied by biopsy and histopathological evaluation, which is primarily based on morphological tissue features. These approaches are inaccurate or inefficient. Our long-term objective is to transform pre- and early epithelial cancer diagnosis through the use of functional metabolic, morphological and biomechanical tissue biomarkers that are extracted non-invasively, automatically and in near real time from fiber-probe-based endogenous two-photon images. Endogenous two-photon imaging is uniquely capable to provide label-free, functional, high resolution tissue images. In this proposal, we aim to establish and validate such measurements and biomarkers for the detection of human cervical pre-cancers using freshly excised tissues. The cervix is an ideal organ for developing and testing our approach as it relaxes some of the size limitations presented for endoscopic applications, enabling us to focus on demonstrating the principles of this innovative platform. In addition, we expect that our proposed methods will enable significant improvements in the specificity of detection of cervical pre-cancers. To achieve our goals, we will acquire images from fifty freshly excised human cervical tissue specimens from patients undergoing colposcopy, loop electrosurgical excision procedure, or hysterectomy. We will acquire endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images and extract signals attributed to NADH, FAD, and collagen. We will process these images using methods we developed to assess: a) the depth-dependent optical redox ratio, mitochondrial organization and nuclear to cytoplasmic ratio variations from FAD and NADH TPEF images of the epithelium, and b) the collagen fiber organization and crosslinking from SHG and TPEF images of the stroma. We will use discriminant analysis to develop algorithms that include the optimal combination of the extracted optical parameters to distinguish high- grade from non-high grade cervical lesions, relying on histopathology as the gold standard. Our algorithms will be entirely automated and fast and will yield a diagnosis based on functional tissue characteristics. Thus, we expect that results from this study will motivate the development of a probe-based 2P imaging system for clinical in vivo imaging translation to enable real-time, highly accurate detection of cervical pre-cancerous lesions. Ultimately, we anticipate that probe-based 2P imaging will transform early cancer diagnosis for a wide range of tissues, such as the oral cavity, the esophagus, the colon, and the bladder.