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To calculate the lifetime and PO2, each image was passed through a twodimensional (2D) smoothing filter, and the background image (at 2500 µs) was subtracted. For each pixel element, the integral from the prescribed delay time to infinity of the function of intensity versus delay time was constructed as follows: I0τ exp(−td /τ), where I0 is the intensity at time zero in arbitrary units, td is the delay time in microseconds, and τ is the lifetime in microseconds.57 This technique requires the injection of a new generation of porphyrin dyes, which have not yet been approved for human use.

4.1.3. Spectrographic Method

Schweitzer used all the wavelengths, but the complete spectrum approach did not produce images based on these wavelengths.58 He used a spectrograph capable of detecting the whole spectrum from a slit-image of the retina. The imaging ophthalmospectrometer was based on a modified fundus camera (RCM 250, Carl Zeiss, Jena, Germany). However, the spectrograph (CP 200, Jobin-Yvon, Longjumeau, France) and intensified CCD matrix camera (576S/RB, Princeton Instruments, Trenton, NJ, USA) were adapted to the ophthalmoscope. A conventional xenon flash lamp illuminated the fundus camera and a slit-like field stop can be switched into the illuminating beam. The illuminated field was confocally placed onto the entrance slit of the spectrograph. The influence of the intraocular scattered light was reduced by the confocal imaging principle. The light coming from the fundus was spectrally dispersed within the spectrograph, and the intensified CCD matrix camera detects a complete reflectance spectrum at the exit plane of the spectrograph in each line. The optical spectrum taken from the vessel blood column was used to determine oxygen saturation using curve fitting methods.58

4.1.4. Three Wavelength Method

Delori employed a three-wavelength spectrophotometric model to measure oxygen saturation in the artery and vein in the retina. The threewavelength method developed by Pittman and Duling and advanced by Zheng et al.59−61 to measure the percentage of oxyhemoglobin in the microvascular system was expanded by Delori to use in the retina. The

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method is based on the difference in the light absorption of oxygenated and deoxygenated hemoglobin. The specific extinction coefficient is given by16

In these formulas, Eλ is the specific extinction coefficient for a mixture of HbO2 and Hb (cm2/µmole), Eo,λ is the specific extinction coefficient of HbO2 (cm2/µmole), Er,λ is the specific extinction coefficient of Hb (cm2/µmole), and λ ≡ Eo,λ − Er,λ. Then, Delori defined the optical density (OD) as follows:

where Dλ is the OD, C the total hemoglobin concentration (µmole/cm3), d the path length (cm), S and s wavelength independent parameters that describe the light scattering by red blood cells.

Dλ can be measured, and it is equal to the negative logarithm of the intensity of reflection at the vessel divided by the intensity of the reflection of the background using

By measuring the OD with at least three wavelengths, Delori solved unknown parameters such as sCd and O2sat.16

The techniques of Delori and Schweitzer are highly innovative and measure the blood oxygen saturation in single retinal vessels. Drawing from these works, a next step was to determine oxygen saturation from all vessels contained in images of the funds. A new method combining spectroscopy and imaging was needed.

4.1.5. Dual Wavelength Technique Using OD Ratio (ODR)

Hickam et al. first employed two wavelengths using photographic film recordings to measure retinal vessel saturation.17 Beach pioneered a twowavelength method for digital oximetry in human subjects, which has been adopted by commercial imaging companies. The ratio of the ODs of blood

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at two wavelengths was shown to bear an approximately linear relationship to hemoglobin oxygen saturation.14,62 Laing et al. later showed that a linear relationship held over the physiological range of oxygen saturation in their falling rabbit paper.19 In the digital method, two high-resolution digital images of the retinal vessels were obtained simultaneously with a single pulse of light. The longer of the wavelengths, at 600 nm, was sensitive to oxygen saturation, while the shorter, at 569 nm, was recorded at a wavelength in which oxyand deoxyhemoglobins had equal light absorbance. Vessel ODs were found for both wavelengths with vessel-tracking algorithms that used the light reflected outside the vessel to estimate incident light on the vessel (Iout), and light reflected from inside vessels (Iin), as a measure of the amount of light absorption in the blood63,64:

ODvessel = log10(Iout/Iin).

At 600 nm, OD was strongly dependent on the amount of oxyhemoglobin present, while at 569 nm, the OD was independent of saturation. Thus, the ODR provided a determination for blood oxygen saturation that compensated for the differences in vessel diameter and small amounts of misfocus that occurred in retinal images. The ODR was determined from the ratio of the vessel ODs found over the same vessel segments in the image: ODR = OD600/OD569.

With this analysis, the ODR varied linearly with blood oxygen saturation measured by pulse oximetry. In Fig. 4.1, the ODR was plotted against systemic blood oxygen saturation for seven subjects. The relationship between the ODR and saturation was offset by different degrees across subjects, and some of the slopes differed significantly from the mean.

The results were partly explained by differing degrees of retinal pigmentation in subjects. The retinal pigment density, which varies across individuals, can affect the light returned from outside the vessels, mostly at the shorter wavelength, and, thus, a pigment correction was added by employing the longer wavelength, where the retinal pigment influence is small, to estimate incident light for vessels in both images. To apply this correction, the relative light throughput for each imaging channel had to be known. This relationship was characterized using the ratio (η) of recorded light intensity at each wavelength that was returned from a diffuse reflecting surface. A pigment-corrected OD was, thus, found for

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Fig. 4.1. ODRs from retinal arteries vs. systemic SO2 (seven subjects). ORD obtained from direct calculation of vessel OD by using method I. Dashed lines, line fits through points from each subject: solid line, line obtained by averaging slope and intercept from all subjects: 000787 +/− 0.00098 (SE). Reprinted with permission from Beach, J.M., Schwenzer, K.J., Srinivas, S., Kim, D., and Tiedeman, J.S. Oximetry of retinal vessels by dual-wavelength imaging: calibration and influence of pigmentation. J Appl Physiol 86:748–758, 1999.

the oxygen-insensitive image and used to find the pigment corrected ODR:

ODcor,569 = log 10 (ηIout600/Iin569), ODRcor = OD600/ODcor,569.

The corrected ODR was plotted against the pulse oximeter reading below for the same subjects (Fig. 4.2):

The correction reduced the coefficient of variation for the ODR across subjects by approximately two-fold. The ODR method reproducibly measured saturation in the larger firstand second-order retinal arteries and veins. Calibration was done empirically with linear fits between artery ODRs and pulse oximetry using inhalation of different concentrations of oxygen. Jim Tiedeman and colleagues in a study of diabetic patients without background retinopathy used an early version of the oximeter.13 This study showed that the retinal vein oxygen saturation was reduced during acute hyperglycemia, and the degree of reduction was greater in

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Fig. 4.2. ODRs from retinal arteries vs. systemic SO2 (seven subjects). ODR corrected for retinal pigmentation ODRcor by using method II. Dashed lines, line fits through points from each subject; solid line, line obtained by averaging slope and intercept from all subjects. 0.00504 +/− 0.00029 (SE) for method II. Reprinted with permission from Beach, J.M., Schwenzer, K.J., Srinivas, S., Kim, D., and Tiedeman, J.S. Oximetry of retinal vessels by dual-wavelength imaging: calibration and influence of pigmentation. J Appl Physiol 86:748–758, 1999.

patients with longer disease duration, suggesting that the autoregulatory response to high sugar becomes impaired. One explanation is that inner retinal oxygen consumption is increased by high levels of sugar, and in the disease condition, autoregulatory responses are not able to increase blood flow to meet demand. Beach et al. collaborated in further developments, which produced an automated retinal oximeter that could identify the saturation in individual vessels without user interaction.64 Specialized software automatically selects measurement points on the oximetry images and calculates the OD (absorbance) of retinal vessels at two wavelengths. Stefansson et al. have applied the automated technique to clinical and vision studies.64,65 Martin Hammer and colleagues have done similar research. Hammer’s method is of special significance and he describes it as follows.66

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“The calculation of the oxygen saturation SO2 was adopted from Beach et al.14 but slightly modified to:

“where Iin and Iout are the intensities of reflected light inside and outside a vessel at the indexed wavelengths measured as gray values in the respective images and η is the ratio of the intensities measured at an ideal white reflector (Spectralon, Labsphere Inc., North Sutton, NH) at 548 nm and 610 nm. In Equation [(4.5)], ODRa,100 is an offset which was introduced as the ODR at the arteriole during inhalation of pure oxygen by Beach et al.,”14 “but was set constant and determined experimentally here. OS is the oxygen sensitivity, another constant to be determined. Since we found linear dependences of the SO2 — value on the vessel diameter as well as on the fundus pigmentation (see results section), we introduced compensation terms into the oximetry equation finally resulting in

“Here, VD is the diameter of the vessel in microns, measured from the image,67 and

“represents the fundus pigmentation by melanin, which extinction decreases approximately linear with the wavelength in the considered spectral range”.68 “The constants a, b, c, and d were determined experimentally from measurements in twenty healthy volunteers (see below). User-friendly software, easy to operate by clinicians and researchers, comprises the following algorithm: In an image, obtained by the camera and filter assembly described as the oximeter above, the operator has to mark the vessel of interest by a mouse click. The vessel is traced automatically; the vessel walls are located as photometric edges in the green channel image, and the vessel is segmented according to changes of its direction at the fundus. For each vessel segment, the pixels inside and outside the vessel are determined by an

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