**2.1. Overview of hypoxia sensing and imaging technologies**

Oxygen-sensitive microelectrodes have long been considered the gold standard for measurement of PO2 in tissues including the retina [2, 56–60]. While this technique gives an accurate and direct measurement of retinal tissue PO2 , there are many drawbacks, most importantly the highly invasive nature of the measurement, as it requires a direct puncture of the retinal tissue which prevents its use in clinics. Furthermore, oxygen imbalances in retinal vascular diseases such as DR originate largely from capillary occlusion and local changes in the retinal vasculature. These occlusions are likely to create small areas of regional hypoxia rather than an entirely hypoxic retinal tissue. Since oxygen sensitive microelectrodes provide point measurements that depend on the placement of the electrode this method will likely miss areas of focal hypoxia that are surrounded by large areas of normoxic tissue unless multiple measurements are made.

Other methods such as MRI have been used to provide insight into oxygen distribution within the retina and are less invasive than oxygen-sensitive microelectrodes. The advantages of MRI are that it is minimally invasive, offers a large field of view, and has no depth limitation. These MRI techniques are often based on the blood oxygenation level-dependent (BOLD) contrast that was first described by Ogawa et al. which rely on natural differences in MR signal between deoxygenated hemoglobin versus oxygenated hemoglobin [61–63], but can also utilize exogenous contrast agents for increased sensitivity as seen in Dynamic Contrast-Enhanced MRI (DCE-MRI) [64]. The use of MRI to detect changes in oxygen levels was first used in the brain but has since been adapted to visualize oxygen fluctuations in the diabetic retina. Berkowitz et al. have used MRI to show increases in blood retinal barrier permeability in rats after 8 months of diabetes [64] and changes in retinal oxygenation in galactosemia-induced diabetic-like retinopathy [43]. The primary limitation of MRI is that the information is typically displayed as either a cross section of the eye or single slice heat maps which are then pieced together to give an overview of the retina [43, 64–67]. This severely limits the techniques ability to provide the adequate resolution required to identify small regions of focal hypoxia in the diabetic retina.

Laser Doppler is another method that has been established to determine blood flow within the retinal vasculature but does not provide information on oxygen PO2 within the retinal vasculature or tissue [68–70]. More recently however, a number of minimally invasive techniques have been established and adapted to measure oxygen tension optically and identify areas of regional hypoxia in the retina. These techniques take full advantage of the unique anatomy of the eye, which unlike other organs is readily accessible and easy to image due to the naturally transparent front of the eye. Methods such as dual wavelength and full spectral retinal oximetry, and also phosphorescence lifetime imaging all provide information on oxygen levels in the retinal vasculature. Other techniques such as hypoxia sensitive fluorescent probes can help to image hypoxic regions within the retinal tissue itself. These techniques are prime candidates for use in a clinical setting due to their minimally invasive nature and their ability to detect areas of focal hypoxia in the diseased retina.

This review will examine the advantages and disadvantages of the imaging techniques that have emerged as potential diagnostic tools for early detection of DR.
