Ophthalmic Time-resolved Confocal Scanning Microfluorometer

项目摘要 这项名为眼科时间分辨共聚焦扫描显微荧光仪(OTR-CSMF)的项目旨在 建立一台能够进行深度分辨荧光光谱的专用共焦显微荧光仪 用于重建生理参数(ph值、O2等)图的角膜。在第一阶段结束时， 该仪器将能够在整个角膜上进行强度和荧光寿命测量 在不同深度，以高深度分辨率(&lt；7微米)持续数小时。该仪器将基于两个 已建立的技术：(1)共焦扫描显微荧光计[由印第安纳州的斯里尼瓦斯博士开发 大学(IU)]，以及(Ii)数字频域技术(DFD)[由ISS开发和营销 Inc.]。OTR-CSMF是一种独特的仪器，能够测量整个角膜深度的荧光 具有很高的深度分辨率。它已被应用于多种情况下研究角膜穿透动力学。 荧光团、角膜生理学和标记纳米颗粒。DFD技术，为荧光而开发 寿命测量，已经非常成熟，并在ISS Inc.销售的光谱仪中进行常规配置。 该第一阶段项目的目标是建立CSMF-DFD系统的全功能原型，并使 这是第一次有一种能够通过角膜进行荧光寿命测量的创新设备。 CSMF-DFD系统的原型将通过以下步骤进行开发：(1)集成CSMF 之前在印第安纳大学使用ISS Inc.的DFD技术的硬件开发成了一个 紧凑型单元。重铸国际空间站正在使用的数据采集和分析软件(VINCI) 利用CSMF-DFD系统，可使用荧光分光光度计实时确定深度分辨寿命。 (2)然后将使用原型CSMF-DFD系统来验证其基于测量的准确性 邻菲咯啉荧光素寿命(寿命~4 ns)和PO2敏感纳米颗粒 (Ru(Phen)3；寿命约300 ns-5微米S)。最后，我们将对深度进行初步研究- 兔角膜体外分辨寿命测量确定经角膜氧分压分布及相关参数 荧光素在组织中的结合。这些目标建立在成功的可行性研究的基础上 ISS Inc.和伊利诺伊大学的斯里尼瓦斯博士在几个月的时间里。 一旦CSMF-DFD系统完全开发，它将允许前所未有的荧光寿命 在体外对角膜进行测量。在开发的下一阶段中的后续修改将 使其能够应用于小动物(活体)，并将测量范围扩大到眼睛的前段。 总体而言，CSMF-DFD系统的可获得性将刺激新的应用来揭开 角膜(如上皮功能障碍、圆锥角膜和Fuchs营养不良)和前段。因此， 该仪器不仅加强了角膜疾病病理生理学的基础科学，而且 也将对药物发现产生直接的翻译影响。

Project Summary This project, titled Ophthalmic Time-resolved Confocal Scanning Microfluorometer (OTR-CSMF), is aimed at building a dedicated confocal microfluorometer capable of depth-resolved fluorescence spectroscopy across the cornea for reconstruction of a map of physiological parameters (ph, O2, etc.). At the end of Phase I stage, the instrument will be able to perform both intensity and fluorescence lifetime measurements across the cornea at different depths with a high depth resolution (< 7 µm) for several hours. The instrument will be based on two established technologies: (i) Confocal scanning microfluorometer (CSMF) [developed by Dr. Srinivas at Indiana University (IU)], and (ii) Digital Frequency Domain technology (DFD) [developed and being marketed by ISS Inc.]. OTR-CSMF is a unique instrument capable of measuring fluorescence across the depth of the cornea with a high depth resolution. It has been applied in several contexts to investigate transcorneal kinetics of fluorophores, corneal physiology, and labeled nanoparticles. The DFD technology, developed for fluorescence lifetime measurements, is highly matured and is routinely configured in spectrometers being sold by ISS Inc. The goal of this Phase I project is to establish a fully functional prototype of the CSMF-DFD system and enable for the first time an innovative device capable of trans-corneal fluorescence lifetime measurements. The prototype of the CSMF-DFD system will be developed through the following steps: (1) Integrate the CSMF previously developed at Indiana University with the hardware of the DFD technology of ISS Inc. into a single compact unit. Recast the data acquisition and analysis software (Vinci) being used in the ISS spectrofluorometers to enable real-time determination of depth-resolved lifetime with the CSMF-DFD system. (2) The prototype CSMF-DFD system will then be employed to verify its accuracy based on measurements of lifetime of fluorescein (lifetime ~ 4 ns) and pO2-sensitive nanoparticles of ruthenium phenanthroline (Ru(Phen)3; lifetime ~ 300 ns - 5 µs) in a microfluidic chamber. Finally, we will perform a pilot study of depth- resolved lifetime measurements in rabbit corneas ex vivo to determine the transcorneal pO2 profile and relative binding of fluorescein across the tissue. These goals are based on successful feasibility studies conducted by ISS Inc. and Dr. Srinivas of IU over several months. Once the CSMF-DFD system is fully developed, it will permit unprecedented fluorescence lifetime measurements across the cornea ex vivo. Subsequent modifications in the next phase of the development will enable its applications to small animals (in vivo) and extend measurements to the anterior segment of the eye. Overall, the availability of the CSMF-DFD system will spur novel applications to unravel the pathophysiology of the cornea (e.g., epithelial dysfunction, keratoconus, and Fuchs dystrophy) and the anterior segment. Thus, the instrument not only enhances the basic sciences underlying pathophysiology of the corneal diseases but also will have a direct translational impact on drug discovery.