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Rebecca M McQuaid, Michael C Mrochen, Ruslan Dmitriev, Dmitri Papkovski, Brian Vohnsen; Monitoring of O2 concentrations during Corneal Cross-Linking (CXL) by Phosphorescence Lifetime Imaging Microscopy (PLIM). Invest. Ophthalmol. Vis. Sci. 2016;57(12):2884.
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© ARVO (1962-2015); The Authors (2016-present)
The diffusion of riboflavin and oxygen is vital for efficient corneal cross-linking (CXL) with UV light. Previous studies found the biomechanical effect of CXL to be oxygen dependent. The purpose of this study is to investigate the role of O2 and its distribution across the stroma before and during CXL through the use of phosphorescence based probes and imaging.
Porcine eyes were obtained from the local slaughterhouse 4 hours post mortem and kept at a temperature of 4oC. The epithelium was removed and the cornea was stained with a solution containing 0.5% riboflavin and infra-red emitting nanoparticles O2 probe for 30 min to allow diffusion. The globe was then analysed at 37° C and 21% ambient O2 on the confocal upright PLIM microscope (Zeiss, Becker & Hickl GmbH) using 5x/0.25 Fluar objective, excitation at 488 nm and emission collected at 750-810 nm. The cornea was imaged over 10 minutes at depths of 0, 50, 100, 150 and 200 µm. The cross-linking was achieved through periodic 20-30 cycles illumination of cornea with UV-A LED light (7 mW/cm2) whilst imaging. Photon distributions and phosphorescence decay curves were analysed after measurement, from which lifetime values and O2 concentrations were calculated and presented as 2D and 3D maps.
We optimised staining with the O2 probe and measurement conditions for the cornea, and performed proof-of-principle PLIM experiments before and after CXL. We observed efficient and uniform in-depth staining of the cornea allowing us to generate high-resolution O2 maps and monitor O2 dynamics during CXL. Previous PLIM results scanning in the Z-direction revealed little to no change in lifetime decays during UV illumination, suggesting axial scanning may be a quicker and more efficient method in quantifying O2 lifetimes during the CXL process.
The use of phosphorescent O2 probes allows for efficient and a minimally-invasive method in measuring O2 prior to, and during CXL. 2D and 3D maps of O2 concentrations across the stroma during CXL will enable us to better understand the role of oxygen during CXL. Future work will focus on measurements under different O2 environments to verify the CXL effect with O2 probes, and to investigate the suitability of O2 PLIM method for future in-vivo use.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Nanoparticle staining of the cornea at depths up to 250µm (ambient conditions only)
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