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

描述（由申请人提供）：许多类型癌症的治疗标准，包括乳腺2-4和甲状腺5、6的恶性肿瘤，是切除肿瘤和周围组织。术后使用常规组织学对切除组织进行评估；然而，这需要处理、包埋、切片和染色最多延迟一天。当手术治疗取决于病理结果时，手术和病理评估之间的延迟会造成并发症。例如，保乳治疗和放疗是早期乳腺癌的标准治疗方法，因为它可以达到与乳房切除术相当的生存率和局部复发率，同时提高术后生活质量8-12。不幸的是，由于大体检查的灵敏度有限，高达40%的病例在手术边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，在切除甲状腺乳头状肿瘤时，如果在肺叶切除术后发现血管或囊被侵犯，通常需要进行第二次手术进行全甲状腺或近全甲状腺切除术。因此，需要影像学技术来实时评估肿瘤病理。作为传统组织病理学的潜在替代品，手术标本的双光子显微镜（TPM）使用内源性或外源性对比保留了传统组织病理学的高分辨率和分子特异性25,26,37。荧光寿命成像显微镜（FLIM）是一种相关的成像方式，可以使用TPM29实现。采用荧光寿命成像（TPM- flim）的TPM通过测量受代谢活性、氧化还原比等参数影响的发射双光子荧光的时间分布，提供了比传统TPM更大的分子对比度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。本建议强调癌症病理实时成像新技术的发展。我们将首先开发一个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拼接（目标1），以高速（3000万像素/秒）成像非常大的领域（>cm2）。然后，我们将开发一个集成系统，用于高速（bbb100 - 1000万像素/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Aim 2中，我们建议在病理实验室对手术标本（丢弃的和临床诊断不需要的）进行研究，开发TPM和TPM- flim，用于实时成像和检测乳腺癌和甲状腺癌病理。本研究将确定可用于检测癌症病理的生物标志物和内在对比来源。最后将TPM-FLIM的诊断性能与金标准进行比较，组织病理学在盲法评估中确定敏感性和特异性。如果成功，这种成像技术最终可以应用于需要实时病理评估的术中应用。

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.