Ocular oximetry, in which blood oxygen saturation is evaluated in retinal tissues, is a promising technique for the prevention, diagnosis and management of many diseases and conditions. However, the development of new tools for evaluating oxygen saturation in the eye fundus has often been limited by the lack of reference tools or techniques for such measurements. In this study, we describe a two-step validation method. The impact of scattering, blood volume fraction and lens yellowing on the oximetry model is investigated using a tissue phantom, while a Monte Carlo model of the light propagation in the eye fundus is used to study the effect of the fundus layered-structure. With this method, we were able to assess the performance of an ocular oximetry technique in the presence of confounding factors and to quantify the impact of the choroidal circulation on the accuracy of the measurements. The presented strategy will be useful to anyone involved in studies based on the eye fundus diffuse reflectance.

Significance: The assessment of biomarkers in the eye is rapidly gaining traction for the screening, diagnosis and monitoring of ocular and neurological diseases. Targeted ocular spectroscopy is a new technology that enables the user to concurrently image the eye fundus and acquire high quality spectra from a targeted region - 1.5 degrees - within the imaged area. The combination of imaging and high-sensitivity spectroscopy provides structural, compositional, and functional information of selected regions of the eye fundus. This opens the door to new, non-invasive approaches to the detection of biomarkers in the eye. Aim: The aim of this study was to demonstrate the multi-modal functionality and validation of the targeted ocular spectroscopy developed. This was done in vitro, using a reference target and a model eye, and in vivo. Approach: Images and spectra from different regions of a reference target and a model eye were acquired and analyzed to validate the system. The same eye model was used to obtain fluorescence images and spectra, highlighting the capability of the system to also perform targeted ocular fluorescence spectroscopy. Subsequently, in vivo imaging and diffuse reflectance spectra were acquired to assess blood oxygen saturation in the optic nerve head and the parafovea of healthy subjects. Results: Tests conducted with the reference target showed that spectral analysis could be accurately performed within specific areas of the imaging space. Moving to the model eye, distinct spectral signatures were observed for the targeted spectral analysis done in the optic disc, the retina and the macula, consistent with the variations in tissue composition and functions between these regions mimicked by the model eye. Further, it was shown that the targeted spectral analysis could also be performed in a fluorescence mode to distinguish various fluorophores present within the imaging space. Finally, in vivo ocular oximetry experiments performed in the optic nerve head and parafovea of healthy patients showed significant differences in blood oxygen saturation between these regions (p = 0.004). Conclusions: Enabling non-invasive, sensitive diffuse reflectance and fluorescence spectroscopy in specific regions of the eye fundus opens the door to a whole new range of monitoring and diagnostic capabilities, from assessment of oxygenation in glaucoma and diabetic retinopathy to photo-oxidation and photo-degradation in age-related macular degeneration.

This study examined the sensitivity of broadband spectroscopy algorithms for retinal tissue oximetry to spectral acquisition parameters. Monte Carlo simulations were conducted on a 4-layer retinal model to assess the impact of various parameters. The optimal spectral range for accurate measurements was determined to be 530 nm to 585 nm. Decreased spectral resolution below 4 nm significantly reduced accuracy. Using an acquisition area larger than the blood vessel resulted in an underestimation of oxygen saturation, especially for high values. A threshold was observed where increased light intensity had no significant impact on measurement variability. The study highlights the importance of informed parameter selection for accurately assessing retinal microcapillary oxygenation and studying local hemodynamics.