Nonlinear Optical Endomicroscopy for Optical Biopsy of Cancer in Internal Organs

描述(申请人提供)：临床上迫切需要一种非侵入性的高分辨率成像技术，用于早期癌症检测和指导内脏器官的活检。双光子荧光(TPF)和二次谐波(SHG)显微技术是解决上述临床需求的一种强有力的技术，它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，而不需要去除组织或外部荧光剂。然而，由于缺乏微型技术平台，其在体内的临床应用仍然极其有限。这一多学科计划的目标是开发一种全光纤扫描内窥镜技术，能够将TPF/SHG显微镜应用于临床进行内脏成像。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。该技术将把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)的柔性光纤探头中，内置飞秒脉冲传输、色散管理、非线性效应抑制、光束聚焦、快速2D光栅束扫描、TPF/SHG采集和焦点跟踪(或深度扫描)机制。小尺寸允许它与标准的危险信号技术(如胃镜)集成在一起。在这项提案中，我们计划解决开发这种内窥镜技术的主要挑战，并评估其用于亚细胞分辨率成像和癌症检测的可行性。其具体目标是：(1)开发新的双包层光纤(DCF)，其为纯二氧化硅芯、大内包层和数值孔径，以显著抑制光纤中的TPF/SHG背景(例如，提高50倍)，并提高TPF/SHG收集效率(例如，比商用DCFF提高约15倍)；(2)开发直径2.1 mm、0.6NA的超消色差微透镜，以将TPF/SHG收集效率提高至少20倍；(3)探索一种基于高阶模式DCFS的新方法，以抑制光纤中的非线性效应，提高TPF/SHG的激发概率；(4)研制占地面积小(1.6×1.6 mm)、驱动电压极低(10V Max)的新型MEMS扫描器，以实现快速二维光栅束扫描和实时聚焦跟踪。将开发一种配备定制的DCFS、微透镜和MEMS扫描仪的全集成内窥镜，能够进行3D TPF/SHG成像；(5)对猪食道进行体内TPF/SHG成像，以评估扫描探头的性能、设计、工程和操作问题；以及(6)使用体外人食道标本评估拟议的癌症检测和肿瘤边缘识别技术的可行性，并将TPF/SHG内窥镜图像与相应的组织学相关联。这项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，并使组织病理的非侵入性实时可视化能够显著提高诊断和活检产量。此外，拟议的内窥镜技术也将适用于许多其他临床方案(尽管不在本提案的范围内)，例如用于指导外科干预和活体组织代谢功能的体内评估。 公共卫生相关性：这项多学科建议的目标是开发和测试一种全光纤3D扫描内窥镜技术，该技术可以将功能强大的台式TPF/SHG显微镜技术应用于临床实践，用于对内脏(如胃肠道)进行成像，这是TPF/SHG显微镜以前无法实现的。这项拟议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化/代谢信息，而不需要切除组织或外部荧光剂，从而实现“光学活组织检查”的形式。这项拟议研究的成功完成将为我们带来一种新的工具，它可以实现组织病理学的原位可视化，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.