The retina is a highly metabolically active tissue, requiring adequate availability of oxygen and other metabolites to generate energy for cellular survival and visual function. Reduction in retinal oxygenation has been implicated in several common retinal diseases, such as vascular occlusions, diabetic retinopathy, glaucoma and retinopathy of prematurity (Alarcon-Martinez et al., 2023; Cringle and Yu, 2010; Dugan and Green, 1991; Mozaffarieh et al., 2008; Ramsey and Arden, 2015; Stefansson, 1990; Yoneya et al., 2002; Zhang et al., 2003). Indeed, a common treatment for combating retinal pathologies and improving vision targets the upregulation of vascular endothelium growth factor (VEGF) due to hypoxia. Under hypoxic condition, one study has shown the level of reduced oxygen tension (PO2) near mitochondria determines whether processes are activated for cell survival or apoptotic cell death (Snyder and Chandel, 2009). Therefore, it is essential to gain knowledge of retinal oxygenation for understanding disease pathophysiology, identifying physiological biomarkers to monitor disease progression and evaluate therapeutic interventions.

Measurements of retinal vascular oxygen saturation (SO2) based on optical absorption spectroscopy of hemoglobin have been reported primarily in large retinal vessels by spectrophotometric oximetry (Hardarson, 2013; Hardarson et al., 2006; Shughoury et al., 2020; Stefánsson et al., 2017), photoacoustic imaging (Hariri et al., 2018; Liu et al., 2013; Song et al., 2012; Zhang et al., 2006), and more recently using visible light optical coherence tomography (Chong et al., 2015; Rubinoff et al., 2023; Song et al., 2020; Wang et al., 2022, 2025; Yi et al., 2015). Retinal venous SO2 and arteriovenous SO2 difference were shown to be potential markers of the ability of the tissue to extract oxygen. Direct measurements of retinal tissue PO2 (tPO2) have been reported in animals using oxygen-sensitive microelectrodes (Lau and Linsenmeier, 2012; Linsenmeier et al., 2023; Linsenmeier and Yancey, 1989; Linsenmeier and Zhang, 2017; Pournaras et al., 1989; Yu and Cringle, 2001). Though potentially invasive to the retinal microenvironment, the oxygen microelectrode technique has superior depth resolution for generating quantitative tPO2 profiles across the retinal depth. This advantage has allowed measurements of outer and inner retinal oxygen consumption (Cringle et al., 2002; Lau and Linsenmeier, 2012; Linsenmeier and Zhang, 2017; Yu and Cringle, 2001).

Phosphorescence lifetime imaging is a technique that measures oxygen levels in living tissues based on oxygen-dependent quenching of phosphorescence (Vanderkooi et al., 1987). After excitation, the oxygen-sensitive phosphor makes a transition from a triplet excited state to ground state, emitting phosphorescence light with a signature lifetime. Since the presence of oxygen quenches the phosphorescence, PO2 can be quantitated by the measurement of phosphorescence lifetime. There are two approaches for measuring the lifetime of phosphorescence emission. With the time-domain approach, lifetime is measured from the exponential decay of intensity as a function of time, thus requiring short pulses of high energy light for excitation with a potential for tissue damage. In contrast, lifetime by the frequency-domain approach is determined based on the phase delay between the modulated incident light and emission, hence is independent of the intensity of the excitation light. Using phosphorescence lifetime imaging, PO2 was measured in the retinal and optic nerve vasculatures of cats and mice using time-domain and frequency-domain approaches (Shonat and Kight, 2003; Shonat et al., 1992). Moreover, measurement of intravascular PO2 at the optic nerve head was reported in minipigs under different light-adapted conditions. (Cranstoun et al., 1997). By combining optical section imaging with phosphorescence lifetime imaging, depth-resolved measurements of retinal vascular and tissue PO2 (vPO2 and tPO2) were demonstrated (Shahidi et al., 2006).