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

在侵袭前阶段检测癌变方面的改进有可能使 对大多数癌症患者的预后和治疗有重大影响。尽管我们在这方面取得了重大进展 对癌症病理生物学的了解，目前最流行的诊断方法仍依赖于低 放大组织成像，然后进行活检和组织病理学检查。这一过程是侵入性的，通常是有限的。 在敏感性和/或特异性方面，耗时长，严重依赖训练有素的专业人员 医生，他们反过来主要利用形态组织的变化来进行评估。这些限制 造成有效治疗的障碍，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)和形态改变上皮癌前期和早期诊断 非侵入性、自动、近乎实时地从光纤探头中提取的组织生物标志物 内源双光子(2P)图像。在这项建议中，我们的目标是建立和验证这种测量和 活体检测人类宫颈癌前病变的生物标志物。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 建议的方法将在短期内显著提高检测的灵敏度和特异度 在阴道镜检查和分诊治疗中发现宫颈癌前病变。为了实现我们的目标，我们有 在学术界和临床同仁之间建立了牢固的伙伴关系，利用 多支队伍。具体地说，我们将利用我们在激光开发(徐，康奈尔)和多光子方面的经验 成像探头设计(Ben-Yakar，UT Austin)，以开发基于探头的2P成像系统，该系统便于携带和 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)。这个 最终的设计规范将进行优化，以实现对图像的自动化、接近实时的分析，从而提供 代谢功能和形态的量化指标(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，和b) 与核质面积比随深度变化有关的形态指标 上皮厚度(Georgakoudi，Tuft)。实时算法将使用新切除的 正常和癌前人类宫颈组织(目标2)。它们将在体内进行测试和进一步优化，当 创新的2P成像平台将用于执行第一次基于人体2P探测器的成像 阴道镜检查(Thieu/Genega，塔夫茨医学中心)(Aim3)。我们预计这些研究将提供高度的敏感性。 和宫颈癌前病变检测的特异性，并将使有可能改变的进一步研究成为可能 可通过探头获得的多种癌症的诊断和最终预后范例。

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.