Ophthalmic Time-resolved Confocal Scanning Microfluorometer

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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.