2.1.

Specular Vessel Glint

A primary concern in the ROx-3 optical system is attenuation of the specular vessel glint. When a small spot of laser light is directed onto a blood vessel in the eye, there are many possible paths it can traverse.^{26, 27, 28, 29, 30} Two paths of particular interest for this discussion are: (i) light specularly reflected from the blood vessel (also known as a glint) and (ii) laterally scattered light that has interacted with the red blood cell–packaged hemoglobin in the vessel and scattered back out of the eye. Figure 1a shows an example of the specular vessel glint path and Fig. 1b shows several examples of laterally scattered rays.

Fig. 1

Two light paths of interest when a retinal vessel is illuminated by a converging cone of light: (a) The glint, which reflects directly from the surface of the vessel and conveys little information about the oxygen saturation, and (b) laterally backscattered light that has interacted with red blood cells.

The glint is a specular reflection from the surface of the vessel. Because this light has not interacted with the hemoglobin inside the vessel, it does not contain information regarding oxygen saturation. This specular reflection frequently appears as a bright line down the center of vessels in retinal images.

The laterally backscattered light is the portion of the return signal that contains useful information about SO₂ (specifically “oxyhemoglobin” saturation, because we are looking at the spectral shift between oxyhemoglobin and deoxyhemoglobin). When a beam is incident on the retinal vessel, some of the light propagates into the blood vessel and interacts with hemoglobin inside the red blood cells, scattering in all directions. Some of this laterally scattered light exits the eye. If multiple monochromatic beams are sent into the eye, then the relative spectrum of this backscattered return signal is highly dependent on the oxygen saturation of the hemoglobin in the red blood cells.^{25, 26} This signal may be many orders of magnitude dimmer than the specular reflection, making it difficult to efficiently collect the light containing oximetric information while rejecting the glint.

The ROx-3 was designed and built to selectively capture light scattered by red blood cells using on-axis illumination, a field stop, and an anticonfocal wire bisecting the field stop. This modified scanning laser ophthalmoscope improves on an earlier design by our group^{17, 20, 31} by using mirrors rather than lenses to remove ghost reflections and an argon-ion multiline laser to generate the discrete wavelengths needed for BGO in this configuration. The illumination wavelength selection and image analysis exhibit the BGO technique.

2.2.

Retinal Oximeter (ROx-3)

The ROx-3, optical layout shown in Fig. 2, is a modified confocal scanning laser ophthalmoscope that uses five monochromatic beams from a multiwavelength argon-ion laser (457.9, 476.5, 488, 496.5, and 514.5 nm) to sequentially scan the retina. The use of a single laser simplifies the system, and this wavelength range is sufficient for measuring SO₂ of 0–100% using the BGO minima technique.^{1, 4} An acousto-optic tunable filter (AOTF) (Crystal Technology AOTF PCAOM NI VIS 55–150 MHz) uses the timing of the fast-scan mirror (FSM) to sequentially switch line by line through each wavelength. A 1-mm-diam pinhole (P) following the AOTF ensures that no residual light from undesired wavelengths enters the system, and a set of adjustment lenses (L1, L2) ensures that the light is collimated. Four concave spherical mirrors (CM1–CM4) in an afocal arrangement direct the beam to the eye, where it is focused on the retina.

Fig. 2

Schematic of the ROx-3.

The light that scatters back out of the eye travels the illumination path in reverse. The portion transmitted by the pellicle beam splitter (BS) (Thorlabs BP208) is imaged onto the field stop-wire filter (FSWF) and detected by a photomultiplier tube (PMT) (Hamamatsu HC 125–01). The 8:92 (8% reflection, 92% transmission) pellicle beam splitter directs the majority of the return light to the PMT. Figure 3 shows how the confocal filter rejects the majority of the specular vessel glint and collects the backscattered light. The confocal filter is a 240-μm wire centered on a 2-mm-diam field stop placed at an image plane (at which the magnification is ∼10), just in front of the PMT. The material around the field stop rejects unwanted stray light coming from the optical system, the anterior chamber of the eye, and the vitreous humor. The central blocking wire rejects much of the light directly reflected back into the collection pathway by the structures anterior to the blood column at the scan site. The light that returns to the detector through the filter is primarily multiply scattered light from the blood within the vessel.

Fig. 3

Field stop-wire filter designed to reject the central vessel glint and transmit the backscattered light that contains hemoglobin information.

A two-dimensional image is generated by confocal scan mirrors and captured by a frame grabber (Foresight Imaging I-50 HSN). The laser beam is raster scanned over part of the retina by a FSM scanning vertically, and a slow-scan mirror (SSM) scanning horizontally. The scan mirrors descan the light returning out of the eye, which is focused onto the confocal filter. The concave mirrors CM1–CM4 image both scan mirrors onto the pupil. This allows the beam to be scanned across the retina without vignetting by using the iris as the pivot for the scans. The frame-grabber board uses sync pulses from the scan mirrors to generate an image with 10-bit depth resolution. The SSM controller triggers the start of each frame at a rate of 10 Hz. A full image is captured every half-cycle, in 50 ms using the FSM for bidirectional scanning. The FSM controller simultaneously triggers the AOTF wavelength switching at the start and end of each row of pixels (a switching rate of 8 kHz), creating an interlaced multiwavelength image. The data analysis relies on comparing the return signal at different wavelengths to determine the oxygen saturation. This technique results in a slight shift between corresponding lines at each wavelength, but at these rates, only a small fraction of images must be discarded due to saccades or vessel motion. The area scanned by the ophthalmoscope is easily adjusted by increasing the angle of the scan mirrors. The device is designed to create images of a (3×3)-mm portion of the retina during targeting (10-deg arc). During measurements, the scan area is decreased to 0.5×0.5 mm (1.68-deg arc). The movement along the retina from line to line at the center of the image when in measurement mode is ∼0.625 μm or 3.75 μm for a complete five-wavelength line set with blank (0.013-deg arc). Two preliminary tests have been performed to assess the functionality of the ROx-3: one on an enucleated swine eye and a second on a human eye.

2.3.

Testing the ROx-3 on an Enucleated Swine Eye

Enucleated swine eyes, with their easily observable blood vessels, are a convenient sample for testing an oximeter.³² They are inexpensive, readily available, and provide a good model of the human eye.³³ They are also useful for preliminary testing before performing live swine or human eye studies with their requirements for a team of doctors, technicians, and associated costs. The living retina continues to remove oxygen from the vessels for several hours after enucleation, making the saturation of the hemoglobin in these vessels near 0%.³⁴

An enucleated swine eye was obtained from the University of Arizona Meat Laboratory. Immediately on pickup, the eye was immersed in a balanced saline solution (BSS) of pH 7.4 and set on ice. Throughout the experiment, the cornea was periodically washed with the BSS to prevent opacification. In order to secure the eye for scanning without distorting its shape, the eye was mounted in a Styrofoam holder using cyanoacrylate and aligned until a retinal vessel was visible, and images were captured with the oximeter.

2.4.

Testing the ROx-3 on a Human Eye

After successful scanning of the enucleated swine eye, scans were made of the eye of a healthy adult volunteer. The laser power at each wavelength was measured with a calibrated silicon detector to ensure that it was <30 μW. This power level has been shown to be within the safety limit for Class I laser devices, as defined by ANSI Z136.1 “Safe Use of Lasers.”³⁵ The subject aligned his head in the proper position, an image was acquired with the ROx-3, and the subject was allowed to move his head (during which time the laser was not directed into his eye). This acquisition process was repeated three times. The experimental protocol was approved by the University of Arizona Institutional Review Board.

2.5.

Data Analysis

Though there is a change in instrument geometry, we are fundamentally collecting the same light information as that gathered via intravitreal illumination^{1, 26} because we are employing the FSWF to block the central glint. Therefore, we can employ the same analysis techniques. The approximations for light paths involved in retinal oximetry described by Denninghoff lead to the simplified equation for data analysis that is used here,¹

BGO is based on the shape of the relative optical density spectrum of hemoglobin³⁸ and is independent of hematocrit and vessel size.¹ Relative ODs were calculated from the ROx-3 images by

Using the model empirically verified by Denninghoff ¹ to further verify this methodology, the measured optical density data with respect to wavelength are expected to fit a line of the form

The calibration line relating the λ_min to SO₂ calculated for swine retinal arteries using intravitreal illumination by Denninghoff ¹ was

We use this intravitreal illumination calibration line to roughly estimate the SO₂ work; however, a new calibration line is needed to properly evaluate the SO₂ measurements. However, the only change in the line is expected to be the intercept,¹ indicating that the [TeX:] ${\rm SEM}_{{\% \rm SO}_{\rm 2} }$ ${\mathrm{SEM}}_{{\%\mathrm{SO}}_2}$ as calculated above is the true error in the SO₂ measurement.

4.1.

On-Axis Blue-Green Oximetry and Removal of the Retinal Vessel Glint

Much of our efforts to provide calibrated blood hemoglobin spectroscopic measurements from the retina using on-axis illumination of the eye has been focused on finding ways to capture multiply scattered light from the retinal vessels while at the same time rejecting glints. The central glint does not appear in the swine images shown in Fig. 4 nor in the swine and human vessel profiles shown in Fig. 5, demonstrating that on-axis illumination can be used with a confocal field stop-wire filter to collect backscattered light and reject the specular vessel glint.

Our prior work showed that background, light path length, blood hemoglobin concentration, and other variations in measurements were independent of wavelength in the spectral range used for BGO when the glint was removed using invasive off-axis illumination.^{1, 26} Similarly, the optical light filtration used in the ROx-3 appears to overcome these challenges.

4.2.

Oxygen Saturation Error

The uncertainty in determining SO₂ can be estimated by using the uncertainty in λ_min and the observation that the error in the linear relationship between λ_min, and SO₂ for all three geometries and samples determined previously has been 3% saturation per 1 nm.^{1, 4, 26, 39} Using this relationship, the maximum error in λ_min from our human vein data of ±0.8 nm corresponds to ±2.3% oxyhemoglobin saturation. The uncertainty of the measurements made using the ROx-3 is in the range we measured elsewhere and what we need for testing the potential use of a retinal oximeter clinically.

4.3.

Backscattering of Light from the Eye in the Blue-Green Spectral Range

In our earlier work evaluating the use of the BGO technique in hemoglobin solutions^{4, 40} and blood in transmission,^{1, 26} we calculated the scattering component of the loss of light using the model equation developed by Steinke and Sheppard.³⁸ The scattering component was linear with wavelength, had a negative slope, and had a positive intercept.⁴ Using a similar approach, we calculated the apparent path length through the blood and the scattering component for each wavelength. We found that the scattering was now a gain (negative intercept) with a positive slope. This is to be expected because the return light would be zero if there was no backscattering in the eye.