In vivo oximetry of human bulbar conjunctival and episcleral microvasculature using snapshot multispectral imaging

Multispectral imaging (MSI) is now well established for non-invasive oximetry of retinal blood vessels, contributing to the understanding of a variety of conditions affecting the retinal circulation, including glaucoma, diabetes, vessel occlusion, and auto-regulation. We report the application of a unique snapshot MSI technique to enable the first oximetric imaging of the blood vessels of the anterior segment, i.e. the episcleral and bulbar conjunctival microvasculature. As well as providing a new capability of oximetry of the scleral vasculature, this technique represents ocular oximetry that is complimentary or alternative to retinal oximetry. We report the oxygen dynamics of these microvascular beds and assess how acute mild hypoxia effects the blood oxygen saturation (SO2) of bulbar conjunctival and episcleral microvasculature. A retinal-fundus camera fitted with a custom Image-Replicating Imaging Spectrometer enabled oximetric imaging of bulbar conjunctival and episcleral microvasculature in ten healthy human subjects at normoxia (21% Fraction of Inspired Oxygen [FiO2]) and acute mild-hypoxia conditions (15% FiO2). Eyelid closure was used to block oxygen diffusion between ambient air and the sclera surface. Four of the ten subjects – those that presented suitable vasculature for direct comparison between bulbar conjunctival and episcleral vessels - were imaged for 30 s following eyelid opening. Vessel diameter and Optical Density Ratio (ODR: a direct proxy for oxygen saturation) of vessels was computed automatically. Oximetry capability was validated using a simple phantom for the scleral vasculature. Average episcleral diameter increased from 78.9 ± 8.7 μm (mean ± standard deviation) at normoxia to 97.6 ± 14.3 μm at hypoxia (p = 0.02). Diameters of bulbar conjunctival vessels showed no significant change from 80.1 ± 7.6 μm at normoxia to 80.6 ± 7.0 μm at hypoxia (p = 0.89). Acute mild hypoxia resulted in a decrease in SO2 (i.e. an increase in ODR) from normoxia levels in both bulbar conjunctival (p < 0.001) and episcleral vessels (p = 0.03). Hypoxic bulbar conjunctival vasculature rapidly re-oxygenated in an exponential manner, reaching normoxia baseline levels, with an average ½ time to full reoxygenation of 3.4 ± 1.4 s. This reoxygenation occurs because the bulbar conjunctival vessels are in direct contact with ambient air. This is the first study to characterise and also to image the oxygen dynamics of bulbar conjunctival and episcleral microvasculature, and to directly observe the rapid reoxygenation of hypoxic bulbar conjunctival vessels when exposed to air. Oxygen diffusion into the bulbar conjunctiva must be taken into account to provide meaningful oximetry because bulbar conjunctival vessels will be highly oxygenated (close to 100% SO2) when exposed to ambient air. Oximetry of bulbar conjunctival vessels could potentially provide insight into conditions where oxygen dynamics of the microvasculature are not fully understood, such as diabetes, sickle-cell diseases, and dry-eye syndrome. Further, in vivo oximetry of individual capillaries and groups of flowing red blood cells could be achieved with a high magnification slit lamp adapted for MSI.