Validity of Retinal Oxygen Saturation Analysis: Hyperspectral Imaging in Visible Wavelength with Fundus Camera and Liquid Crystal Wavelength Tunable Filter Yoko HIROHARA 1;2 , Yoshitaka OKAWA 1 , Toshifumi MIHASHI 1;2 , Tatsuo YAMAGUCHI 2 , Naoki NAKAZAWA 2 , Yasuko TSURUGA 2 , Hiroyuki AOKI 2 , Naoyuki MAEDA 3 , Ichiro UCHIDA 4 , and Takashi FUJIKADO 1 1 Department of Applied Visual Science, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan 2 Research Institute, Topcon Corporation, Tokyo 174-8580, Japan 3 Department of Ophthalmology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan 4 Department of Anesthesiology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan (Received September 7, 2006; Accepted March 8, 2007) The purpose of this paper was to investigate the feasibility of a newly developed hyperspectral fundus imaging camera with a liquid crystal tunable filter. The intensities of different wavelengths of light transmitted through an artery, vein, and the area surrounding these vessels and reflected out were measured, and the differential spectral absorptions were analyzed. Measurements were made from 16 normal eyes and from two artificial capillaries. The ratios of absorption (ROA) of arteries to veins from 500 to 580 nm (range 1) and from 600 to 720 nm (range 2) were calculated. For all eyes, the ROA range1 was larger than ROA range2 . The ROA obtained from the artificial capillary filled with blood saturated with oxygen or nitrogen was similar to that of simulated data of oxy- and deoxyhemoglobin extinction rate. Most ROAs of human eyes were lower than those of the simulated data and the artificial capillaries. Oxygen saturation analysis by hyperspectral fundus imaging of retinal vessels were qualitatively in agreement with the in vitro analysis or simulated values. However, further improvements are necessary to evaluate the oxygen saturation quantitatively in the retinal blood vessels. # 2007 The Optical Society of Japan Key words: hyperspectral imaging, absorption, optical density, oxygen saturation, liquid crystal wavelength tunable filter 1. Introduction The measurement of the oxygen saturation in blood is based on the principle that the extinction properties of oxy- and deoxyhemoglobin (HbO 2 , Hb) are different in the visible and infrared spectral range. Because of this difference, spectral imaging has been used to investigate the oxygen saturation of the blood in vessels of the brain and skin. 1,2) For the eyes, the oxygen saturation in the retinal blood vessels has been estimated. 3–6) Thus, Dinn et al. analyzed oxygen saturation using a new method with an innovative optical density calculation using eight bit digital image and suggested that the new method could use for retinal oximetry, 4) while Yoneya et al. analyzed oxygen saturation in patients with central retinal vein occlusion (CRVO) using Fourier transform-based spectral retinal imaging and showed the oxygen saturation level was correlated with the severity of CRVO. 5) Delori developed a retinal vessel oximeter using a scanning fundus camera to measure optical density of a retinal vessel, 6) and Khoobehi et al. evaluated oxygen saturation in the retinal vessels using a prism-grating-prism architecture and found that pure oxygen increased oxygen saturation in veins and increasing intraocular pressures reduced oxygen saturation in the vessels. 7) Recently, hyperspectral or multispectral imaging has been used in various fields. One of the novel components of this imaging system is a liquid crystal wavelength tunable filter (LCF). Hyperspectral imaging with a LCF has advantages over the regular charge-coupled device (CCD) camera or other methods of spectral measurements in that a direct representation of two-dimensional images and a detailed spectral analysis with various wavelengths is possible. Although many studies have been reported on the oxygen saturation of retinal blood, none has ever been widely used in the clinic. 8) The reasons for this may be that the accuracy of the measurements is not sufficient to be useful for clinical use. The purpose of this study was to determine the validity of using absorption analyses by hyperspectral imaging to determine the oxygen saturation in the blood of retinal vessels. To accomplish this, images of the retina and artificial capillaries filled with blood were obtained by the hyperspectral fundus camera, and the spectral absorption properties of the blood were analyzed. 2. Materials and Methods 2.1 Human subjects Sixteen normal eyes of 16 normal subjects whose average  standard deviation (SD) ages was 25:9  6:1 year were studied. The procedures used were approved by the Osaka University Institutional Review Board, and a written informed consent was obtained from all subjects after an explanation of the purpose and procedures to be used. All procedures conformed to the tenets of the Declaration of Helsinki. 2.2 Artificial capillaries and circulated blood Glass tubings were filled with blood to simulate artificial  E-mail address: fujikado@ophthal.med.osaka-u.ac.jp OPTICAL REVIEW Vol. 14, No. 3 (2007) 151–158 151