Two-Photon Fluorescence Lifetime Microscopy for Breast and Thyroid Cancer Margin

描述(申请人提供)：许多类型的癌症，包括乳房恶性肿瘤2-4和甲状腺5，6的标准护理是肿瘤和周围组织的切除。切除的组织在术后使用传统的组织学方法进行评估；然而，这需要长达一天的处理、包埋、切片和染色7。手术和病理评估之间的这种延迟造成了手术治疗依赖于病理结果时的并发症。例如，保乳治疗和放射治疗是早期乳腺癌的标准护理，因为它实现了与乳房切除术相同的存活率和局部复发率，同时提供了更好的术后生活质量8-12。不幸的是，由于肉眼检查的敏感性有限，在高达40%的病例16、17中，残余肿瘤出现在手术边缘附近，因此患者经常需要再次手术。类似地，在切除乳头状甲状腺肿瘤时，叶切除后发现血管或包膜侵犯，通常需要二次手术来进行全或接近全甲状腺切除术。因此，需要成像技术来实时评估癌症的病理。作为传统组织病理学的一种潜在替代方法，手术标本的双光子显微镜(TPM)保留了使用内源性或外源性对照25、26和37的传统组织病理学的高分辨率和分子特异性。荧光寿命成像显微镜(FLIM)是一种可以使用TPM29实现的相关成像方式。具有荧光寿命成像的TPM(TPM-FLIM)通过测量受代谢活性、氧化还原比和其他参数30、33、35、36影响的发射的双光子荧光的时间分布，提供比传统TPM显著更大的分子对比度。与癌症相关的代谢变化已被证明改变了几种常见的荧光细胞代谢物的寿命33。这项建议强调开发癌症病理实时成像的新技术。我们将首先开发一种TPM系统，能够使用快速光学扫描、超短脉冲激励和样品镶嵌(目标1)以高速(&gt；3000万像素/S)对非常大的视场(&gt；1 cm2)进行成像。然后，我们将开发一个用于高速(&gt；1-1000万像素/S)3D TPM-Flim全组织成像的集成系统，该系统使用基于光学时钟数字化仪的新型光学采样技术。在目标2中，我们建议在病理学实验室对手术标本(被丢弃的和不需要临床诊断的)进行研究，以开发用于乳腺癌和甲状腺癌病理的实时成像和检测的TPM和TPM-FLIM。这项研究将确定可用于检测癌症病理的生物标记物和内在对比来源。最后将TPM-FLIM的诊断性能与金标准进行比较，在组织病理学中进行盲法评估，以确定敏感性和特异性。如果成功，这项成像技术最终可以应用于需要实时病理评估的一系列术中应用。

DESCRIPTION (provided by applicant): The standard of care for many types of cancer, including malignant tumors of the breast2-4 and thyroid5, 6, is resection of the tumor and surrounding tissue. Excised tissue is evaluated postoperatively using conventional histology; however this requires a delay of up to one day for processing, embedding, sectioning and staining7. This delay between surgery and pathological assessment poses complications when surgical treatment depends on pathology findings. For example, breast-conserving therapy and radiation is a standard of care in early breast cancer because it achieves survival and local recurrence rates equivalent to those of mastectomy while providing improved post-operative quality of life8-12. Unfortunately because of the limited sensitivity of gross examination, residua tumor is present near the surgical margin in up to 40% of cases16, 17 and patients therefore often require repeat surgeries. Similarly, in the resection of papillary thyroid neoplasm, a findin of vascular or capsular invasion following a lobectomy typically requires a second surgery to perform a total or near-total thyroidectomy6. Therefore, there is a need for imaging technology to assess cancer pathology in real-time. A potential alternative to conventional histopathology, two photon microscopy (TPM) of surgical specimens retains the high resolution and molecular specificity of conventional histopathology using endogenous or exogenous contrast25, 26, and 37. Fluorescent lifetime imaging microscopy (FLIM) is a related imaging modality that can be implemented using TPM29. TPM with fluorescence lifetime imaging (TPM-FLIM) provides significantly greater molecular contrast then conventional TPM by measuring the temporal profile of emitted two-photon fluorescence which is influenced by metabolic activity, redox ratio and other parameters30, 33, 35, 36. Changes in metabolism associated with cancer have been shown to alter the lifetimes of several common fluorescent cellular metabolites33. This proposal emphasizes the development of new technology for real-time imaging of cancer pathology. We will first develop a TPM system capable of imaging very large fields (>1 cm2) at high speed (>30 megapixels/s) using a fast optical scanning, an ultrashort pulse excitation and sample mosaicking (Aim 1). We will then develop an integrated system for high-speed (>1-10 megapixel/s) 3D TPM-FLIM whole tissue imaging using a novel optical sampling technique based on an optically clocked digitizer. In Aim 2, we propose to conduct a study of surgical specimens (discarded and not required for clinical diagnosis) in the pathology laboratory to develop both TPM and TPM-FLIM for real-time imaging and detection of breast and thyroid cancer pathologies. This study will identify biomarkers and sources of intrinsic contrast that can be used to detect cancer pathology. Finally the diagnostic performance of TPM-FLIM will be compared to the gold standard, histopathology in a blinded evaluation to determine sensitivity and specificity. If successful, this imaging technology could ultimately be applied to a range of intraoperative applications where real-time assessment of pathology is required.