Real time imaging of the aqueous outflow pathway by two-photon endoscopy

描述（由申请人提供）：众所周知，原发性开角型青光眼的眼内压升高与房水流出阻力增加有关。然而，阻力的确切位置和房水流出受损的机制仍然不清楚。即使在正常眼中，房水流出阻力的部位也没有被精确定位。为了解决这些问题，需要新的成像技术。双光子激光扫描显微镜是一种很有前途的成像技术，与传统技术相比具有许多优点，包括更高的扫描速率和更低的光毒性。此外，双光子激光扫描显微镜具有能够对组织内深处的结构进行成像而不会引起失焦漂白的显著优势。这种显微技术的分析适用于观察器官的三维组织。然而，它的应用仅限于那些可以很容易地固定在普通显微镜载物台上的盖玻片上的受试者。最近已经尝试开发小型化的显微内窥镜。具有小型化探针的双光子内窥镜系统已经实现了对位于体腔内和/或难以放置在常规显微镜载物台上的各种组织（例如大脑）的真实的时间成像。需要进一步改进以使用这种内窥镜系统来对眼睛中的流出路径进行真实的实时活细胞/结构成像。特别是，微探针/扫描系统应该是防水的，并且，特别是对于离体（ab interno）实验，探针应该以防水的方式安装到灌注眼前节培养物中。我们建议建立一个新的双光子内窥镜，是特定的眼睛，能够研究活细胞/结构的流出途径。该系统将由包括飞秒激发激光器、激光传输单元、荧光检测单元、成像采集单元和新的内窥镜探头/扫描系统在内的组件构建。由于了解流出途径需要活细胞/结构的真实的时间成像，因此双光子内窥镜系统将用于离体灌注人眼前节培养以及猴眼的体内研究。为了从内部（从前房侧）观察人体流出通道的活细胞/结构，将双光子内窥镜系统安装在离体灌注式人体眼前节培养系统上，并建立真实的时间活细胞/结构成像。对于房水流出通路的体内成像，将观察猴眼，并从外部（从眼睛外部）观察房水流出通路，以在不干扰前房的情况下对其成像。这将在巩膜瓣被移除后尝试，有或没有移除施累姆氏管的外壁。活组织的黄绿素染色将是实验设计的一部分。这些研究工作的结果可能会导致新的真实的时间成像方法调查的流出途径，并将有助于说明水流出的路线和事件。 公共卫生相关性：通常与青光眼相关的眼内压升高与眼内房水排出减少有关。目前的技术很难观察到房水流出的引流途径;因此，一种新的成像系统，如双光子激光扫描显微镜，将更好地了解该系统，这可能会导致青光眼的新治疗方法。该项目的目标是开发一种新的内窥镜系统，该系统专为研究眼睛而设计，具有出色的分辨率和扫描速度，从而可以实现活体流出通道的双光子成像。

DESCRIPTION (provided by applicant): It is well known that the elevated intraocular pressure of primary open angle glaucoma is related to an increased resistance to the outflow of aqueous humor. The precise location of the resistance and the mechanisms by which aqueous humor outflow is impaired, however, are still not clear. Even in normal eyes, the locus of aqueous humor outflow resistance has not been pinpointed. In order to resolve these questions, novel imaging techniques are needed. Two-photon laser scanning microscopy is a promising imaging technique that has many advantages over conventional techniques, including higher scanning rate and lower phototoxicity. In addition, two-photon laser scanning microscopy has the significant advantage of being able to image structures deep within a tissue without incurring out-of-focus bleaching. Analyses from this microscopic technique are appropriate for observing the three-dimensional organization of organs. Its application, however, is limited to subjects that can be easily immobilized and apposed to a coverglass on the stage of a regular microscope. Attempts have recently been made to develop a miniaturized microendoscope. A two-photon endoscope system with a miniaturized probe has enabled real time imaging of various tissues, such as the brain, that sit inside a body cavity and/or are difficult to place on a regular microscope stage. Further improvements are required to use such endoscope systems for real time live cell/structure imaging of the outflow pathway in the eye. In particular, the microprobe/scanning system should be watertight, and, especially for ex vivo (ab interno) experiments, the probe should be mounted into the perfusion anterior segment culture in a watertight fashion. We propose to build a new two-photon endoscope that is specific for the eye and capable of studying live cell/structure of the outflow pathway. The system will be built with components including a femtosecond excitation laser, a laser delivery unit, a fluorescence detection unit, an imaging acquisition unit, and a new endoprobe/scanning system. Because understanding the outflow pathway necessitates real time imaging of live cell/structure, the two-photon endoscopy system will be used in an ex vivo perfusion human anterior segment culture as well as in in vivo studies of monkey eyes. To observe the live cell/structure of the human outflow pathway ab interno (from anterior chamber side), the two-photon endoscopic system will be mounted on an ex vivo perfusion human anterior segment culture system, and real time live cell/structure imaging will be established. For in vivo imaging of the aqueous outflow pathway, monkey eyes will be observed, and the aqueous outflow pathway will be observed ab externo (from outside of the eye) to image it without disturbing the anterior chamber. This will be attempted after scleral flaps have been removed, with and without removal of the outer wall of Schlemm's canal. Fluorescein staining for live tissue will be part of the experimental design. The findings of these research endeavors may lead to novel real time imaging methods for investigating the outflow pathway and will help illustrate the aqueous outflow route and events. PUBLIC HEALTH RELEVANCE: The elevation of intraocular pressure often associated with glaucoma is related to reduced drainage of the aqueous humor fluid from the eye. The drainage pathway of the aqueous outflow is difficult to view with current technology; therefore, a new imaging system, such as two-photon laser scanning microscopy, would provide a better understanding of this system which may lead to newer treatments for glaucoma. The goal of the project is to develop a new endoscope system that is specifically designed for the study of the eye with excellent resolution and scanning speed so that two-photon imaging of the living outflow pathway can be achieved.