Probe-based two photon microscopy for functional, label-free early cancer diagnosis

侵入前阶段癌变检测的改进有可能使 对大多数癌症患者的预后和治疗产生重大影响。尽管我们在这方面取得了重要进展 对癌症病理学的了解，最流行的诊断方法仍然依赖于低水平 放大组织可视化，然后进行活检和组织病理学。这个过程是侵入性的，通常是有限的 由于其敏感性和/或特异性，耗时，并且在很大程度上依赖于训练有素的专业知识 医生反过来主要利用形态组织变化进行评估。这些限制 给有效治疗带来障碍，并增加金钱和心理成本。我们的长期目标是 通过使用功能（代谢）和形态学改变上皮癌前期和早期诊断 从基于光纤探针的非侵入性、自动且近乎实时的方式提取组织生物标志物 内源双光子（2P）图像。在本提案中，我们的目标是建立并验证此类测量和 用于体内检测人类宫颈癌前病变的生物标志物。子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的一些尺寸限制， 使我们能够专注于展示这个创新平台的原则。此外，我们期望我们的 所提出的方法将在近期显着提高检测的灵敏度和特异性 阴道镜检查期间宫颈癌前病变的诊断和分诊治疗。为了实现我们的目标，我们有 利用学术界和临床界的优势和专业知识，在学术界和临床界的同事之间建立了牢固的伙伴关系 多个团队。具体来说，我们将利用我们在激光开发（Xu，康奈尔大学）和多光子方面的经验 成像探针设计（Ben-Yakar，UT Austin）开发基于探针的 2P 成像系统，该系统是便携式的 能够以亚微米分辨率快速采集整个宫颈上皮深度的图像（目标 1）。的 最终设计规范将进行优化，以实现对图像的自动化、近实时分析，从而提供 代谢功能和形态的定量指标（目标 2）。特别是，我们将评估：a) 多个 基于氧化还原比和线粒体组织参数的细胞代谢定量生物标志物 从内源性 NAD(P)H 和 FAD 2P 激发的上皮荧光图像中提取，b) 与核与细胞质面积比的深度依赖性变化相关的形态学指标和 上皮厚度（Georgakoudi，塔夫茨）。将使用新切除的数据建立实时算法 正常和癌前人类宫颈组织（目标 2）。它们将在体内进行测试并进一步优化 创新的 2P 成像平台将用于在人类首次基于 2P 探针的成像 阴道镜检查（Thieu/Genega，塔夫茨医疗中心）（Aim3）。我们预计这些研究将提供高灵敏度 和宫颈癌前检测的特异性，将使进一步的研究有可能改变 可通过探针对多种癌症进行诊断和最终预后的范例。

Improvements in the detection of cancerous changes at the pre-invasive stage have the potential to make a significant impact in the prognosis and treatment of most cancer patients. Despite important advances in our understanding of cancer pathobiology, the most prevalent diagnostic methods continue to rely on low magnification tissue visualization, followed by biopsy and histopathology. This process is invasive, often limited in its sensitivity and/or specificity, time-consuming, and relies heavily on the expertise of highly trained physicians, who in turn exploit primarily morphological tissue changes for their assessments. These limitations present barriers to effective treatment and raise monetary and psychological costs. Our long-term objective is to transform pre- and early epithelial cancer diagnosis through the use of functional (metabolic) and morphological tissue biomarkers that are extracted non-invasively, automatically, and in near real time from fiber-probe-based endogenous two-photon (2P) images. In this proposal, we aim to establish and validate such measurements and biomarkers for the detection of human cervical pre-cancers in vivo. 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 near-term improvements in the sensitivity and specificity of detection of cervical pre-cancerous lesions during colposcopy and triage with therapy. To achieve our goals, we have forged a strong partnership among colleagues in academia and the clinic, leveraging strengths and expertise of multiple teams. Specifically, we will exploit our experience in laser development (Xu, Cornell) and multiphoton imaging probe design (Ben-Yakar, UT Austin) to develop a probe-based 2P imaging system that is portable and enables fast image acquisition throughout the cervical epithelium depth with submicron resolution (Aim 1). The final design specifications will be optimized to enable automated, near-real time analysis of images that provides quantitative metrics of metabolic function and morphology (Aim 2). In particular, we will assess: a) multiple quantitative biomarkers of cellular metabolism based on redox ratio and mitochondrial organization parameters extracted from endogenous NAD(P)H and FAD 2P excited fluorescence images of the epithelium, and b) morphological metrics associated with depth-dependent variations of the nuclear to cytoplasmic area ratio and epithelial thickness (Georgakoudi, Tufts). The real time algorithms will be established using freshly excised normal and pre-cancerous human cervical tissues (Aim 2). They will be tested and further optimized in vivo when the innovative 2P imaging platform will be used to perform the first-in-human 2P probe-based imaging during colposcopy (Thieu/Genega, Tufts Medical Center) (Aim3). We expect these studies will provide high sensitivity and specificity of cervical pre-cancer detection and will enable further studies that have the potential to change the paradigm of diagnosis and ultimately prognosis for a broad range of cancers that are accessible via a probe.