Our method for retinal oximetry is based on the work of Beach et al.
2 The setup and outcome are shown in
Figure 1 .
Two retinal oximeters were used for the study, one based in Indianapolis and the other in Reykjavik. Both are composed of a fundus camera coupled with a beam splitter to a digital camera. (In Indianapolis, the fundus camera was a Topcon TRC50-VT; Topcon Co., Tokyo, Japan; the beam splitter was a MultiSpec Patho-Imager; Optical Insights, Tucson, AZ; and the digital camera was a CoolSNAP ES; Roper Scientific, Tucson, AZ. In Reykjavik, the same beam splitter was used, but the fundus camera was a Canon CR6-45NM; Canon Inc., Tokyo, Japan; the digital camera was an SBIG ST-7E; Santa Barbara Instrument Group, Santa Barbara, CA, used at 2 × 2 binning). The beam splitter separates the original image into four optical channels. In each channel, there is a different narrow band-pass filter, through which only light of specific wavelengths can pass. The center wavelengths of the filters are 542, 558, 586, and 605 nm and the half-bandwidth is 5 nm, except for the 542-nm filter, which has a 9-nm half-bandwidth. In the present study, only the 586- and 605-nm wavelengths were used.
With a specialized computer program, optical density (OD) can be calculated for every vessel at each of the four wavelengths. OD is a measure of the blood’s light absorbance and is calculated as
\[\mathrm{OD}{=}\mathrm{log}(\ \frac{I_{0}}{I}),\]
where
I and
I o are the brightness levels inside and just outside a vessel, respectively. It can be shown that the ratio of ODs at certain wavelengths (OD ratio, ODR) has an inverse and approximately linear relationship to hemoglobin oxygenation
2 4 :
\[\mathrm{SO}_{2}{=}a{+}k{\cdot}(\ \frac{\mathrm{OD}_{X}}{\mathrm{OD}_{Y}}){=}a{+}k{\cdot}\mathrm{ODR}.\]
In
equation 2 , SO
2 is the percentage of hemoglobin oxygen saturation;
a and
k are constants; OD
X and OD
Y are ODs (no unit) at wavelengths
X and
Y, respectively; and ODR is the optical density ratio. Thus, in theory, hemoglobin oxygenation can be calculated using brightness inside and outside vessels at two wavelengths of light.