COMBINED TWO PHOTON OPTICAL COHERENCE MICROSCOPY FOR INTRAVITAL FUNCT IMAGING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 该项目涉及开发一种新型的高分辨率功能成像设备。它将双光子显微镜(TPM)和光学相干层析成像(OCT)结合到一个平台上。这两种方法都利用超短脉冲近红外激光技术，需要光束扫描才能形成图像。然而，OCT中的信息量来自于散射光，而TPM信号来自于荧光的非线性电子激发。它们共同构成了活体显微镜，相当于单个细胞中的相位对比度和荧光覆盖图像。但使用TPM/OCT，可以在0.5-1 mm的组织深度形成断层图像。最近，一个法国小组首次展示了设计一种结合了这两种方法的系统是可能的。我们建议将这项工作进一步推进几个步骤，纳入关键的光学设计元素，以优化我们对癌症相关过程的敏感度。我们的活体功能成像仪将能够同时成像新生血管结构、血流方向/速度、细胞外基质结构(胶原/弹性蛋白纤维)和细胞氧化还原状态(如氧化应激)。这些图像将由本征信号形成，即干涉图和双光子激发的自体荧光，而不需要添加外源染料/探针。然而，该系统也可以与添加的发色团一起使用，如荧光染料和基因表达探针(GFP等)。图像将被实时渲染；因此，只需将受试者放在成像舞台上，就可以在模型肿瘤系统中进行活体动物模型研究。此外，该仪器的结构将使人体皮肤损伤的研究可以在体内进行，目标是开发一种提供可与组织病理学相媲美的信息的技术。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project involves the development of a new type of high-resolution functional imaging device. It combines 2-photon microscopy (TPM) and optical coherence tomography (OCT) into a single platform. These methods both utilize ultrashort-pulse near-infrared laser technologies and require beam scanning to form images. However, the information content in OCT is derived from scattered light, whereas TPM signals originate from non-linear electronic excitation of fluorescence. Together, they constitute the intravital microscope equivalent of a phase contrast and fluorescence overlay image in a single cell. But with TPM/OCT, tomographic images can be formed at depths of 0.5-1 mm in tissue. Recently, a French group demonstrated, for the first time, that it is possible to design a system that combines the 2 approaches. We propose to take this work several steps further by incorporating key optical design elements that will optimize our sensitivity to cancer relevant pro cess es. Our intravital functional imager will be capable of simultaneously imaging neovascular structure, blood flow direction/velocity, extracellular matrix structure (collagen/elastin fibers), and cellular redox state (e.g. oxidative stress). These images will be formed from intrinsic signals, i.e. interferograms and 2-photon excited autofluorescence, without the addition of exogenous dyes/probes. However the system can also be used with added chromophores such as fluorescent dyes and gene expression probes (GFP, etc). Images will be rendered in real time; so live animal model studies can be conducted in model tumor systems simply by placing the subject on an imaging stage. In addition, the instrument will be constructed so that studies of human skin lesions can be performed in vivo, with the goal of developing a technique that provides information comparable to histopathology.