We demonstrated dynamic retinal-vessel analysis of the rat eye for the first time. For this purpose, we developed a simplified rat eye model (reduced schematic rat eye, or rSRE) to be used with ZEMAX, in both sequential and non-sequential mode. The model enabled definition of the optic design requirements prior to design and analysis of a novel non-contact retina camera adapted to the rat eye. Nearly reflex-free fundus imaging was achieved. We created a novel Dynamic Vessel Analyzer for the rat by combining this retina camera with an adapted Xe light source and the standard Dynamic Vessel Analyzer platform.
In dynamic retinal-vessel analysis, the reliability of the measuring procedure was demonstrated in 9 rats with baseline recordings without provocation (). Measurements yielded stable readings for both artery and vein, with coefficient of variation around 1% for the arteries and 0.6% for the veins. The drift of the arterial and venous measurements over the 7-minute period (< 1%) can be considered negligible taking into account the slight fluctuations in blood pressure in an in-vivo experiment.
Considering that pulse amplitude in humans is approximately 1%, these variations are likely caused by biological factors such as pulsation and vasomotion; this assumption is also supported by the fact that variation is higher in arteries, because we would expect pulsation amplitudes to be higher in arteries than in veins. However, we accept the possibility of a slight reading error in these variations due to weaker signal-to-noise ratio (SNR), which would probably be more pronounced in the arteries. Because the current frame rate of 25 fps does not meet the sampling theorem at the heart rate of a rat, a higher frame rate may reveal more details regarding biological influences on the baseline time course.
High-contrast reflex-free fundus images were produced in a pigmented Brown-Norway rat and a non-pigmented Wistar rat (). Compared with the Brown-Norway rat, approximately one-tenth of the light was needed for the Wistar rat because of its higher fundus reflectivity and lack of light-absorbing pigmentation. A residual reflex arising from the ophthalmoscopic lens was present in the Brown-Norway fundus image, but this had no negative effect on retinal imaging or DVA. Eye and retina camera alignment, along with individual vessel selection, are additional strengths of the developed system.
Eye safety was estimated in accordance to the specified fundamental requirements for optical radiation safety for ophthalmic instruments in humans (ISO 15004-2:2007) for continuous-wave operation aware of having no valid damage data for the rat. Light intensity was measured in terms of radiant flux using a research radiometer (IL1700, International Light Technologies, Peabody, Massachusetts, USA) together with a measuring head (GRO-268R, Laser 2000 GmbH, Wessling, Germany). For the Brown-Norway rat we measured 2 mW in the cornea plane. Considering the annular area of the illumination beam we obtained irradiance values of 76 mW/cm2 for the cornea, and with a 32° fundus field, 27 mW/cm2 for the retina. This means a safety factor of 52 for the cornea and 26 for the retina, respectively.
Fundus imaging and the DVA results from and indicate the direct response between the designed and realized opto-mechanical DVA-R setups, and provide conclusive evidence that stop design must be considered first, before correcting for optical aberrations. With the fundus imaging and dynamic vessel analysis (DVA) results from and , it can be resumed that the substitution of all system lenses for standard lenses was sufficient in terms of optical performance for imaging the major retinal vessels and performing DVA. The astigmatism illustrated in and did not have an obvious effect on both, imaging and DVA. The simulation results from enabled assessment of imaging performance including the rat eye optics. If the measured MTF contrast for a rat eye for a 1 mm pupil is 0.5 [34
], neglecting the difference in wavelength (560 nm vs. 632.8 nm), we calculated a factor of approximately 5, in spatial frequency, compared to the system MTF at 0.5 contrast. The pictogram at 5.2 cyc/deg at a contrast of 0.5 in represents the on-axis performance of the new eye model. That means, with the system MTF curves shown, the on-axis modulation transfer through the retina camera optics is nearly diffraction limited, because the system MTF contrast of 0.45 lies just slightly below the model eye contrast of 0.5 at 5.2 cyc/deg (see on-axis MTF curve in , blue solid line). With a contrast of 0.47 at the field height of 0.9613 mm the performance is even better. In terms of the spot diagram, field curvature, and distortion data, the performance parameters () appear acceptable for imaging and measurements of vessel diameter. This is because we selected only a small part of the total field of view; i.e., the part relevant for measurement. In future work, we will attempt to reduce astigmatism, the drifting of sagittal and tangential image planes. Image performance may be enhanced by using customized lenses.
The optical system was designed in reverse order, starting at the rat eye, because the parameter values were clearly defined (). To focus on stop design (and therefore separation of illumination and imaging), we began with a paraxial design and applied the exact data for heights and angles using the corresponding ZEMAX editors (pupil imaging in and object imaging in ). We did not consider any kind of transmission optimization in either the paraxial or lens design (e.g., the use of étendue to target the maximum lumen throughput), but compensated by applying a Xe light source to the low illumination path transmission.
Light–eye interaction is a crucial factor in fundus imaging by indirect ophthalmoscopy, and small eyes pose an additional challenge. The outer-ring diameter is limited by the iris, and the inner-ring diameter by the size of the central imaging aperture. The resulting geometric separation of illumination and imaging paths is necessary for good imaging contrast; any violation leads to impaired image quality. It is previously noted [30
] that the corneal window (CW) is the key in fundus camera design. To determine CW in the rat eye, published schematic rat eye data [33
] were transformed into ZEMAX. But the small spherical inner-core structure (radius of 0.958 mm) prevented its use in ZEMAX sequential mode, for tracing ray bundles sequentially filling the system stop, and for fields larger than 13° half-angle for a 0.25 mm pupil diameter, or 6.5° for a 1 mm pupil diameter. At this point, we introduced a new reduced schematic rat eye suitable for sequential ray tracing in ZEMAX (data listed in ), which consists of fewer surfaces and has the major difference of a higher lens refractive index: 1.665 vs. 1.5 published in [33
]. The higher refractive index is not inconsistent with eye anatomy or Hughes’ core lens model because the total refractive powers are identical. The advantage of the new model is that it enables continuous ray tracing in sequential mode. To define CW, the focus of modeling was the anterior eye, in particular the cornea and the lens, instead of considering a more complex structured eye model; e.g., a gradient index schematic eye [36
]. The differences in effective focal length and refractive power (< 1 dpt), respectively, between the new model and Hughes´ model can be neglected when other influences are taken into account, such as tear film thickness and axial length alterations (1 dpt ≈10 µm difference in effective focal length). Different radii of curvature and refractive indices in the eye, whether schematic or real eye, will evoke slightly different exit angles at the cornea, and result in varying CWs. In practice, this uncertainty is considered by using an aperture stop in the imaging path.
MTF rat eye data were studied for two main reasons. First, it was necessary to find a compromise for the applicable pupil diameter in the rat. Smaller diameters lead to reduced geometric optical aberrations and greater depth of foci, whereas larger diameters lead to higher transmission. A 1 mm diameter was chosen. For that diameter, the model eye performance is similar to in-vivo data reported in [34
]. The wavelength difference can be neglected. Measurements in mice evoked best optical performance roughly at this diameter [29
]. However, all MTF data calculated and discussed above include all optical aberrations and thus represent how rodents naturally see. Analyzed with Shack-Hartmann wavefront sensors and best corrected, better optical performances are possible as measurements in mice show [29
]. Second, prior to the assembling of the experimental setup, it was important to assess the complete system (rat eye model and optics of the retina camera) referring to in-vivo modulation transfer performance (see ).