Ophthalmic Time-resolved Confocal Scanning Microfluorometer

眼科时间分辨共焦扫描显微荧光计

• 批准号: 10080180
• 负责人: Beniamino Barbieri
• 金额: $ 19.48万
• 依托单位: ISS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080180
• 关键词: Affect; Animals; Anterior; Anterior eyeball segment structure; Aqueous Humor; Basic Science; Binding; Biochemical; Biological Assay; Cattle; Characteristics; Communities; Computer software; Confocal Microscopy; Cornea; Corneal Diseases; Data Analyses; Development; Devices; Diffusion; Epithelial; Epithelium; Eye; Family suidae; Feasibility Studies; Fluorescein; Fluorescence; Fluorescence Microscopy; Fluorescence Spectroscopy; Fluorometry; Frequencies; Fuchs' Endothelial Dystrophy; Functional disorder; Goals; Health; Hour; Human; Indiana; Investigation; Keratoconus; Kinetics; Label; Legal patent; Maps; Measurement; Measures; Microfluidics; Modification; Molecular; Ophthalmology; Oryctolagus cuniculus; Outcome; Oxygen; Partial Pressure; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenanthrolines; Photons; Physiological; Physiology; Pilot Projects; Process; Property; Research; Resolution; Retina; Ruthenium; Scanning; Speed; System; Technology; Testing; Time; Tissues; Universities; Validation; Vision; analog; base; biophysical properties; data acquisition; design; digital; drug development; drug discovery; experimental study; extracellular; fluorophore; fundamental research; in vivo; in vivo evaluation; innovation; instrument; millisecond; nanoparticle; novel; operation; phase 1 study; pre-clinical research; prototype; reconstruction; time use; translational impact

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.

项目摘要 该项目名为眼科时间分辨共焦扫描显微荧光计（OTR-CSMF），旨在 建立一个专用的共焦显微荧光计能够深度分辨荧光光谱跨越 用于重建生理参数（ph、O2等）的图的角膜。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 以高深度分辨率（< 7 µm）在不同深度进行数小时的测量。该工具将基于两个 已建立的技术：（i）共焦扫描显微荧光计（CSMF）[由印第安纳州的Srinivas博士开发 （ii）数字频域技术（DFD）[由ISS开发和销售 Inc.]。OTR-CSMF是一种能够测量角膜深度荧光的独特仪器 具有高深度分辨率。它已被应用在几个方面，以研究经角膜动力学的 荧光团、角膜生理学和标记的纳米颗粒。DFD技术，为荧光而开发 寿命测量，是高度成熟的，并常规配置在光谱仪正在出售的ISS公司。 第一阶段项目的目标是建立CSMF-DFD系统的全功能原型， 这是第一次能够进行跨角膜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开发：（1）集成CSMF 先前在印第安纳州大学用ISS公司的DFD技术的硬件开发。成单个 紧凑的单元。改造国际空间站使用的数据采集和分析软件（芬奇软件） 光谱荧光仪，使实时确定的深度分辨寿命与CSMF-DFD系统。 (2)原型CSMF-DFD系统将被用来验证其准确性的基础上测量 钌菲咯啉的荧光素寿命（寿命~ 4 ns）和pO 2敏感纳米颗粒 （Ru（Phen）3;寿命~ 300 ns - 5 µs）。最后，我们将进行一项深入的试点研究- 离体兔角膜的分辨寿命测量，以确定经角膜pO 2分布和相对 荧光素穿过组织的结合。这些目标是基于成功的可行性研究， ISS Inc.和IU的Srinivas博士进行了几个月的合作。 一旦CSMF-DFD系统完全开发，它将允许前所未有的荧光寿命 在离体角膜上的测量。下一阶段开发的后续修改将 使其能够应用于小动物（体内），并将测量扩展到眼前节。 总的来说，CSMF-DFD系统的可用性将刺激新的应用，以揭示疾病的病理生理学。 角膜（例如，上皮功能障碍、圆锥角膜和Fuchs营养不良）和眼前节。因此，在本发明中， 该仪器不仅增强了角膜疾病病理生理学的基础科学， 也将对药物发现产生直接的转化影响。