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

描述（由申请人提供）：许多类型癌症（包括乳腺恶性肿瘤2 -4和甲状腺恶性肿瘤5，6）的标准治疗是切除肿瘤和周围组织。术后使用常规组织学对切除的组织进行评估;然而，这需要延迟长达一天的处理、包埋、切片和染色7。当手术治疗依赖于病理学发现时，手术和病理学评估之间的这种延迟造成并发症。例如，保乳治疗和放疗是早期乳腺癌的标准治疗，因为它实现了与乳房切除术相当的生存率和局部复发率，同时改善了术后生活质量8 -12。不幸的是，由于肉眼检查的敏感性有限，高达40%的病例在手术切缘附近存在残留肿瘤16，17，因此患者通常需要重复手术。同样，在乳头状甲状腺肿瘤切除术中，叶切除术后发现血管或包膜浸润通常需要第二次手术进行甲状腺全切或近全切6。因此，需要成像技术来实时评估癌症病理学。作为传统组织病理学的潜在替代方法，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比剂的传统组织病理学的高分辨率和分子特异性25，26和37。荧光寿命成像显微镜（FLIM）是一种相关的成像方式，可以使用TPM 29实现。TPM与荧光寿命成像（TPM-FLIM）提供了显着更大的分子对比度比传统的TPM通过测量发射的双光子荧光的时间分布，这是受代谢活动，氧化还原比和其他参数的影响30，33，35，36。与癌症相关的代谢变化已被证明会改变几种常见荧光细胞代谢物的寿命33。该提案强调开发用于癌症病理学实时成像的新技术。我们将首先开发一种TPM系统，该系统能够使用快速光学扫描、超短脉冲激发和样品镶嵌（Aim 1）以高速（>30百万像素/秒）成像非常大的场（>1 cm 2）。然后，我们将开发一个集成系统，用于高速（>1-10兆像素/秒）3D TPM-FLIM全组织成像，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中对手术标本（丢弃且不需要用于临床诊断）进行研究，以开发TPM和TPM-FLIM，用于乳腺癌和甲状腺癌病理的实时成像和检测。这项研究将确定可用于检测癌症病理学的生物标志物和内在对比度的来源。最后，将TPM-FLIM的诊断性能与金标准进行比较，在盲法评价中进行组织病理学，以确定灵敏度和特异性。如果成功的话，这种成像技术最终可以应用于一系列需要实时评估病理的术中应用。

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.