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

描述(申请人提供)：众所周知，原发性开角型青光眼眼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。即使在正常眼睛中，房水流出阻力的位置也没有被精确地确定。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扫描速度快、光毒性低等优点。此外，双光子激光扫描显微镜具有显著的优势，能够在不引起焦外漂白的情况下对组织内部的结构进行成像。这种显微技术的分析适合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常规显微镜舞台上的盖子上的对象。最近，人们试图开发一种小型化的显微内窥镜。具有微型探头的双光子内窥镜系统已经能够实时成像各种组织，例如大脑，这些组织位于体腔内和/或难以放置在常规显微镜平台上。需要进一步的改进以使用这种内窥镜系统对眼睛中的流出路径进行实时活细胞/结构成像。特别是，微探针/扫描系统应该是水密的，特别是对于体外(Ab Interno)实验，探头应该以水密的方式安装到灌流前段培养物中。我们建议建立一种新的双光子内窥镜，这种内窥镜专用于眼睛，能够研究流出通道的活细胞/结构。该系统将由飞秒激发激光器、激光发射单元、荧光检测单元、成像采集单元和新的内探头/扫描系统等部件组成。由于了解流出途径需要对活细胞/结构进行实时成像，因此双光子内窥镜系统将用于体外灌流的人眼前节培养以及猴眼的体内研究。将双光子内窥镜系统安装在体外灌流的人眼前段培养系统上，建立实时的活细胞/结构成像系统，以观察(从前房侧)人眼内流出道的活细胞/结构。对于房水流出通路的活体成像，将观察猴眼，并将从外(眼)外观察房水流出通路，以在不干扰前房的情况下对其成像。在摘除巩膜瓣后，在切除Schlemm管外壁和不摘除Schlemm管外壁的情况下，都会尝试这样做。对活组织进行荧光素染色将是实验设计的一部分。这些研究工作的发现可能导致新的实时成像方法来研究流出途径，并将有助于阐明水流出途径和事件。 公共卫生相关性：眼压升高通常与青光眼相关，与眼内房水引流减少有关。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，将提供对这一系统的更好的了解，这可能导致青光眼的新治疗方法。该项目的目标是开发一种专门为眼睛研究而设计的新内窥镜系统，具有出色的分辨率和扫描速度，从而实现对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.