Oxygen supply supports the metabolism of the human retina, and abnormal oxygen perfusion is associated with various retinal conditions leading to vision damage and blindness, including glaucoma, age-related macular degeneration, diabetic retinopathy, and vascular occlusions (1). Therefore, the measurement of blood oxygen saturation within the retinal circulation is not only essential to understanding the physiopathology of retinal diseases, but also can play important role for detection and monitoring.

Label-free optical retinal oximetry harnesses the oxygen-dependent spectral contrast of endogenous hemoglobin to non-invasively measure oxygen saturation quantitatively in the human retina (2–6). A fundus camera based multi-wavelength oximetry has been previously reported in several clinical studies on retinal conditions (7). However, the technique lacks depth resolution, and the measurement is complicated by the reflections from vessel surface. So far, only readings from large vessels in the peri-papillary region have been reported.

Visible light optical coherence tomography (VIS-OCT) is a recent development that provides the necessary 3D imaging capacity to eliminate confounding signals from other retinal layers and the choroid (8–12). The much stronger absorption in visible light range than conventional near infrared light (8), in conjunction with 3D segmentation of retinal blood vessels, permits reliable spatio-spectral analysis for microvascular retinal oximetry (11). By combining OCT angiography, capillary oximetry in the human retina has been recently reported which is a significant technical contribution. The initial report of in vivo oximetry using VIS-OCT was introduced in 2011 (13), then VIS-OCT oximetry was applied in rat's retina in 2013 (8). After several years of development, the technique has been successfully demonstrated in human retina in pathological cases for clinical research, including glaucoma, retinal ischemia, diabetic retinopathy (DR), central retinal vein occlusion (CRVO) and sickle cell retinopathy (SCR) (14–16).

Being responsible for the central vision field and acuity, macular region contains >30% of the total ganglion cells in the whole retina (17) in addition to being enriched with cone photoreceptors. The quantification of macular vascular sO₂ has important implications for macular retinal function and metabolism. However, existing reports so far have evaluated the sO₂ of large retinal vessel around the optic nerve head (ONH) (4, 18–20). Due to lack of resolution in fundus-based 2D oximetry, macular region vascular sO₂ has not been sufficiently studied before. In this study, for the first time, we report a baseline study of sO₂ of parafoveal arterioles and venules. We analyze the association of the arterial and venous sO₂ with ophthalmic exam, and OCT macular and ONH scans. The result will serve as an important reference for the future comparison with pathological data.

Fourteen healthy subjects in total were recruited in this study. Four subjects were excluded due to the failure of fixation, or low image quality. Fixation failures were due to significant eye movements during imaging where fovea was not present in the images, or the vessel locations were not recognizable. Lower image qualities caused the unsuccessful segmentation, leading to the failure of vessel selection and sO₂ calculation. Additional four eyes were excluded by the same criterion within the left ten subjects. The demographic information and the ophthalmic measurements are summarized in Tables 1, 2. Sixteen eyes in total were used for the analysis. Nine eyes have mild Grade 1 cataract.

The sO₂ value for all arterioles and venules were first averaged within each eye, as shown in Figure 2. The mean arterial (AsO₂) and venous sO₂ (VsO₂) were 92.1 ± 7.1 (vol %) and 48.4 ± 5.0 (vol %) respectively. The mean arteriovenous sO₂ difference (A-V sO₂) was 43.8 ± 9.5 (vol %). The mean and standard deviation of vessel sO₂ for both arterioles and venules from all eyes are shown in Supplementary Figure 3.

Table 3 summarizes the correlation coefficients and p values between parafoveal AsO₂, VsO₂, A-V sO₂ and ophthalmic measurements including refractive errors, IOP, thickness of macular ganglion cell complex (GCC) and circumpapillary RNFL (cpRNFL). No significant correlation was found except between VsO₂ and IOP, cup/disk ratio (CDR) and age. Figure 3 shows the correlation matrix map between sO₂ per eye and other ophthalmic parameters.

We further analyzed the correlation between sO₂ and vessel topographic parameters, which were in the Supplementary Figures 1, 2. No significant correlation was found between sO₂ and vessel length (L), area (A), diameter (D), and the distance of ρ_min and ρ_max.

For the venules, A and L is significantly correlated as expected by geometry. The diameter positively correlated with the length and area of venules but with no significance. The minimum distance (ρ_min) has a negative correlation with length, area, and diameter, but only has a significant dependency on the latter two. The maximum distance (ρ_max) has significant dependencies on all the other dimensions except diameter. For the arterioles, sO₂ has no significant correlation with topographic parameters. All the topographic parameters have significant correlation with each other, except for the ρ_min with length, area, and diameter.

Table 4 summarizes the topographic parameters and sO₂ values from all of arterious and venous segments. The AsO₂ and VsO₂ per segments are 91.0 ± 8.0 vol % (n = 57) and 50.9 ± 8.8 vol % (n = 43), and the diameter of venules is significantly larger than the arterioles' (P < 0.001). For the other parameters, no significant difference was found. It is noted that diameters of both arterioles and venules are smaller than 40 μm.

In this paper, we report the first baseline study on parafoveal vessel oxygen saturation (sO₂) in healthy eyes. We quantified the topographic features of those vessels and found no significant correlation with sO₂ values. There is no significant correlation between sO₂ and cpRNFL, GCC thickness or refractive error. IOP moderately correlated with VsO₂; however, A-V sO₂ had no significant correlation with IOP. The significant association between VsO₂ and age and CDR was also found.

We found the negative correlation between VsO₂ and age in healthy subjects was significant, which is consistent with previous reports (24–26). We also found correlation between VsO₂ and IOP. There has been sparse literature investigating the association between VsO₂ and IOP. Liu et al. reported no association between IOP and sO₂ for the healthy children under 18 (27). However, there is a significant age difference in our study and thus it is difficult to compare. It is plausible that IOP was positively correlated to the age of people under 60 (28), leading to the negative correlation to the VsO₂. The positive association between VsO₂ and CDR was marginally significant, which can also be found in Vandewalle et al. (29). Despite above significant correlation, we found no significant correlation between A-V sO₂ with age, IOP or CDR.

Compared to fundus-based or multi wavelength SLO oximetry (7, 30), VIS-OCT provides better depth resolution enabling precise segmentation from the bottom of the vessels, and avoids noise from other layers. The parafoveal vessels in this study have a modest diameter ranging from 20 to 30 μm where visible light may penetrate fully. Without the depth segmentation, the penetrated light would diffuse through other layers and create confounding factors in the fundus camera. This would also compromise the resolution and contrast and make macular oximetry challenging for 2D image-based methods. We also note that the relative smaller caliber of the parafoveal vessels may be better suited for VIS-OCT than larger peripapillary vessels around ONH, since the bottom of the vessels were easier to image than large vessels to create a strong reflectance signal.

While this is the first study of the parafoveal vessel sO₂ in normal human subjects using VIS-OCT oximetry, there are limitations worth noting. First, we had limited subject number in this pilot study. Therefore, we refrained from advanced multivariable statistical analysis and rather performed simple two-sample t-tests and Pearson's linear correlation. Marginal significances were found in some parameters stated above. A larger cohort would be needed in the future study to validate those significance by a multiple-variable analysis to incorporate variable interactions and other confounding factors. Second, there are the technical limitations that we would like to improve in the future. The spectral bandwidth in the current device is limited in exchange for large imaging depth for better alignment. With our new linear-K spectrometer design and extended imaging depth (31), the spectral bandwidth can be increased to improve the robustness and accuracy of sO₂ calculation.

In summary, we investigated parafoveal microvascular sO₂ using VIS-OCT on healthy subjects in this initial baseline study. The statistical analysis reveals that no significant association between sO₂ and vessel topographic features. Also, there are no significant correlations for the eye sO₂ and clinical parameters except the relation of VsO₂ and IOP, CDR and age. This study will provide a normal (an initial) database for later sO₂ investigations in ocular diseases.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Boston Medical Center. The patients/participants provided their written informed consent to participate in this study.

JY supervised this project. JW and JY analyzed the results. JW, WS, MF, MD, and JY contributed the manuscript. WS, NS, MF, and MD assisted in subject screening and recruitment. All authors contributed to the article and approved the submitted version.

NEI/NINDS R01NS108464, R01 EY032163 and R21EY029412.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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