Bahram Khoobehi and James M. Beach

4.4. Experiment Three

4.4.1. Methods and Materials

All methods and materials used in Sec. 4.4 have been described in the above experiment except for the following.

4.4.1.1. Compliance testing

Compliance testing was performed in each monkey eye three times over a three-week period before starting the HSI studies. Briefly, the position of the ONH, termed mean position of the disc or MPD, was measured at normal IOP (10 mmHg) after 10 and 30 minutes (baseline). Then, IOP was acutely increased to 30 mmHg, and the change in MPD (amount of posterior deformation or movement of the ONH) was determined after 10 and 30 minutes. The compliance test images were obtained by a one-time automatic pre-scan with a 670-diode laser at 4–6 mm depth and 15◦ scan angle (Heidelberg Retinal Tomograph-II, Heidelberg Engineering, Heidelberg, Germany). The tomograph software (Heidelberg Eye Explorer) computed and automatically set parameters to acquire three-dimensional (3D) topography images from the images obtained in pre-scan. Digitization was performed in frames of 384 × 384 pixels. After the mean topography image was computed automatically from the three topography images, the optic disc margin was defined manually. Finally, the stereometric parameters were computed automatically and compared with the results of previous examinations.

4.4.1.2. Hyperspectral imaging

Hyperspectral images were obtained as previously described. During each imaging session, IOP in the dilated eye was set at 10 mmHg (normal), and then raised to 30, 45, and 55 mmHg. Three images were taken at each of two time points: 10 and 30 minutes after IOP elevation. To test reproducibility, each session was repeated five times over a 10-week period. Following the completion of the series of HSI imaging sessions on each monkey, compliance tests were repeated. Following data collection from the first eye, the entire test and image protocol were carried out in the other eye. Thus, 150 images were obtained at each IOP and time point (three images/session × five sessions/eye × two eyes/monkey × five monkeys), and 2400 images

158

Hyperspectral Image Analysis for Oxygen Saturation Automated Localization of the Eye

obtained in all (150 images/time point-IOP × two time points/IOP × four IOPs/eye × 2 eyes/monkey).

4.4.1.3. Selection of ONH structures

Distinct regions within the border of the optic disc were obtained manually from one image (569 nm band) taken from the set of HSI images, where good contrast of the vessels enabled image pixels to be grouped into artery and vein structures, and ONH areas, including the nasal and temporal cup and the rim (nasal, temporal, cup, superior and inferior aspects), as shown in Fig. 4.18.

4.4.1.4. Statistical methods

The data were analyzed using a nested analysis of variance (ANOVA).70 In this ANOVA model, the saturation level of oxygen was the outcome, the site of measurement and IOP were the main effects, and the regions of the retina examined constituted the nested arrangement of treatments. Mean arterial

Fig. 4.18. Image of the ONH and overlying retinal vessels obtained at one spectral image (569 nm) of the hyperspectral image set. The areas for calculation of oxygen saturation in each of the seven regions include between 300 and 1000 pixels: A, artery; V, vein; C, cup; T, temporal ONH; N, nasal ONH; S, superior ONH; and I, inferior ONH. Reprinted with permission from Beach, J., Ning, J., and Khoobehi, B. Oxygen saturation in optic nerve head structures by hyperspectral image analysis. Curr Eye Res 32:161–70, 2007. ©2007 Informa Medical and Pharmaceutical Science.

159