Nonlinear Optical Endomicroscopy for Optical Biopsy of Cancer in Internal Organs

描述（由申请人提供）：临床上迫切需要一种无创高分辨率成像技术，用于早期癌症检测和引导内脏器官活检。双光子荧光（TPF）和二次谐波发生（SHG）显微术是一种强大的技术，通过以亚细胞分辨率提供关于生物组织的结构和生化/代谢信息来解决上述临床需求，而不需要组织去除或外部荧光剂。然而，由于缺乏微型技术平台，其体内临床应用仍然非常有限。这个多学科的建议的目的是开发一种全光纤扫描显微内镜技术，能够将TPF/SHG显微镜带到临床内部器官成像。它涉及5个合作伙伴，其中2个来自学术界，3个来自工业界。该技术将扫描激光显微镜的所有基本功能集成到一个小直径（~2.4 - 3.4 mm）的单个柔性光纤探头中，内置飞秒脉冲传输机制，色散管理，非线性效应抑制，光束聚焦，快速2D光栅光束扫描，TPF/SHG收集和焦点跟踪（或深度扫描）。小尺寸允许其与标准红旗技术（如胃镜）集成。在这项提案中，我们计划解决开发这种显微内镜技术的主要挑战，并评估其用于亚细胞分辨率成像和癌症检测的可行性。具体目标是：（1）开发具有纯石英纤芯、大内包层和数值孔径的新型双包层光纤（DCF），以显著抑制光纤内TPF/SHG背景（例如50倍）并提高TPF/SHG收集效率（例如，约15倍）超过市售DCF;（2）研制了直径为2.1mm、NA为0.6的超消色差微透镜，使TPF/SHG的收集效率比GRIN透镜提高至少20倍;（3）探索基于高阶模DCF抑制光纤非线性效应、提高TPF/SHG激发几率的新方法;（4）开发新的小尺寸MEMS扫描仪（1.6 x 1.6 mm）和极低的驱动电压（最大10 V），可实现快速2D光栅光束扫描和实时焦点跟踪。将开发具有定制DCF、微透镜和MEMS扫描器的完全集成的内窥镜，能够进行3D TPF/SHG成像;（5）对猪食管进行体内内窥镜TPF/SHG成像，以评估扫描探针的性能、设计、工程和操作问题;以及（6）使用离体人食管样本评估所提出的用于癌症检测和肿瘤边缘识别的技术的可行性，并将TPF/SHG显微内镜图像与相应的组织学相关联。该研究的意义在于将强大的TPF/SHG显微镜技术转化为临床实践，用于内脏器官的癌症检测和图像引导活检，并实现原位组织病理学的无创实时可视化，以显着提高诊断和活检率。此外，所提出的显微内镜技术也将适用于（尽管超出了本提案的范围）许多其他临床场景，例如用于指导手术干预和活体组织代谢功能的体内评估。 公共卫生相关性：这项多学科提案的目的是开发和测试一种全光纤3D扫描显微内镜技术，该技术可以将强大的台式TPF/SHG显微镜技术应用于临床实践，用于对内脏器官（如胃肠道）进行成像，而这在以前是TPF/SHG显微镜无法实现的。所提出的技术可以通过以亚细胞分辨率提供关于生物组织的结构和生物化学/代谢信息而以"光学活检"的形式起作用，而不需要组织去除或外部荧光剂。拟议研究的成功完成将为我们带来一种新的工具，它可以实现原位组织病理学的可视化，并提高我们早期癌症检测，肿瘤边缘识别以及活检和干预指导的能力。

DESCRIPTION (provided by applicant): There is a critical clinical need for a noninvasive high-resolution imaging technology for early cancer detection and guidance of biopsy in internal organs. Two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy is a powerful technology to address the above clinical need by providing structural and biochemical/metabolic information about biological tissues at subcellular resolution without the need for tissue removal or external fluorescent agents. However, its in vivo clinical application remains extremely limited due to the lack of a miniature technology platform. The objective of this multidisciplinary proposal is to develop an all-fiber-optic scanning endomicroscopy technology which is able to bring TPF/SHG microscopy to clinic for internal organ imaging. It involves 5 partners with 2 from academia and 3 from industry. The proposed technology will integrate all essential functions of a scanning laser microscope into a single flexible fiber-optic probe of a small diameter (~2.4-3.4 mm), with built-in mechanisms for femtosecond pulse delivery, dispersion management, nonlinear effect suppression, beam focusing, rapid 2D raster beam scanning, TPF/SHG collection, and focus tracking (or depth scanning). The small size permits its integration with a standard red-flagging technology (such as a gastroscope). In this proposal, we plan to tackle the major challenges in developing such an endomicroscopy technology and evaluate its feasibility for subcellular resolution imaging and for cancer detection. The Specific Aims are to: (1) Develop new double-clad fibers (DCF) of a pure silica core, large inner clad and numerical aperture to dramatically suppress the in-fiber TPF/SHG background (e.g. by 50 folds) and improve TPF/SHG collection efficiency (e.g. by ~15 folds) over commercially available DCFs; (2) Develop a super-achromatic microlens of a 2.1mm diameter and 0.6 NA to improve the TPF/SHG collection efficiency by at least 20 folds over a GRIN lens; (3) Explore a novel approach based on high-order mode DCFs to suppress nonlinear effects in optic fiber and improve TPF/SHG excitation probability; (4) Develop novel MEMS scanners of a small footprint (1.6 x 1.6 mm) and an extremely low drive voltage (10V max) to achieve rapid 2D raster beam scanning and real-time focus tracking. A fully integrated endomicroscope with customized DCFs, microlens and MEMS scanners will be developed, capable of 3D TPF/SHG imaging; (5) Conduct in vivo endoscopic TPF/SHG imaging of swine esophagus to evaluate the performance, and design, engineering and operation issues of the scanning probe; and (6) Evaluate the feasibility of the proposed technology for cancer detection and tumor margin identification using ex vivo human esophagus specimens, and correlate TPF/SHG endomicroscopy images with corresponding histology. The significance of the proposed research is to translate the powerful TPF/SHG microscopy technology to clinical practice for cancer detection and image-guided biopsy in internal organs, and enable noninvasive real-time visualization of tissue histopathology in situ to significantly improve diagnostic and biopsy yields. In addition, the proposed endomicroscopy technology will also be applicable (although outside the scope of this proposal) to many other clinical scenarios such as for guidance of surgical interventions and for in vivo assessment of metabolic function of living tissues. PUBLIC HEALTH RELEVANCE: The objective of this multidisciplinary proposal is to develop and test an all-fiber-optic 3D scanning endomicroscopy technology, which can bring the powerful bench-top TPF/SHG microscopy technology to clinical practice for imaging internal organs (such as the gastrointestinal tract) that was not previously possible with TPF/SHG microscopy. The proposed technology can function in a form of "optical biopsy" by providing structural and biochemical/metabolic information about biological tissues at subcellular resolution without the need for tissue removal or external fluorescent agents. Successful completion of the proposed research will bring us a new tool, which enables visualization of histopathology in situ and improves our capability for early cancer detection, tumor margin identification, and guidance of biopsy and interventions.