This article presents a method to visualize and analyze the FAZ and parafoveal capillary network noninvasively. The approach is based on the idea of using motion as intrinsic contrast. The key assumption is that blood cells move relative to their surrounding photoreceptors; thus, by extracting motion from videos of the eye, one can visualize perfused vessels. Motion is extracted using multi-frame division videos; using division images enables multiple division images to be averaged together arithmetically. This averaging enhances the final SNR. In general, noninvasive videos of the microcirculation from many imaging modalities suffer from low contrast, a common challenge for many vessel analysis algorithms. Our technique for motion contrast enhancement is general, and can be applied as an enabling step for more advanced analyses, such as quantification of the FAZ and parafoveal capillary density.
There are several advantages to using this approach. Most importantly, it is noninvasive, and can potentially be applied with low risk for subjects ranging from normal to pre-disease to disease states. The comparison of the vessel montage to FA shows that the method is able to detect retinal capillaries and generate a complete parafoveal map. FAs were not performed on other subjects to avoid unnecessary risks associated for eyes with no ocular conditions. Another advantage of this technique is the availability of other retinal measurements from the same dataset. The photoreceptor mosaic can be analyzed from unprocessed videos (, ). Also, it is possible to investigate the speed and pulsatility of individual leukocytes as they move through capillaries, using offline analysis.10,20
Finally, one can extend the analysis to compute statistics such as vessel density, as illustrated in this article.
The main limitations to the method are: (1) it may be too time consuming for immediate clinical use; (2) subjects must have clear ocular media; and (3) good subject compliance is necessary. First, our technical methods for visualization of the parafoveal capillaries were developed to demonstrate a new concept, and not for immediate clinical implementation. Also, the time to acquire overlapping videos may influence which subjects might be good candidates. Second, there are many potential subjects that have clear ocular media, particularly subjects in normal and pre-diseased states, as well as some subjects with certain diseases; these subjects are usually precluded from invasive procedures such as FA. Finally, while good subject compliance is helpful during imaging sessions, the subject tasks are relatively straightforward, involving fixation on targets presented at various locations.
There are tradeoffs when imaging with two different laser wavelengths. A prior study showed that green laser wavelengths were optimal for vessel contrast in scanning laser ophthalmoscopy (SLO) systems.21
While we found that the contrast of vessels and flow through vessels was higher for the 532 nm laser compared with the 840 nm laser (), we did not observe any differences in the vessels that could be identified for analysis. However, there are major advantages to using the 840 nm laser. The SNR of the photoreceptors is much higher for videos acquired at 840 nm, an important consideration when evaluating photoreceptor health; this was because we imposed conservative light exposure limits. To ensure safe light levels, the power that reached the subjects' retinas was maintained at a level that was at 10x below the Maximum Permissible Exposure limit defined by the American National Standards Institute.22
Since the SNR improves as the power of light increases, and since we imposed our conservative light exposure limits, the videos acquired at 532 nm had considerably lower SNRs. Finally, the lower brightness 840 nm light source (~50 trolands) was better for subject comfort.
Despite variations in imaging parameters, we were able to achieve good results overall with our algorithms. Such variation would potentially require variations in video and image processing parameters; however, we were able to apply the same general method to all videos. We also showed that we could achieve similar results using videos from different imaging sessions (). Due to the unique nature of this approach, there are no direct methods to validate the approach other than comparison to FA. Even when contrast agents are combined with SLO,3,23
the level of detail that we achieved was not observed. Previously, the only established method for noninvasive visualization of the FAZ and parafoveal capillary network was based on the entoptic blue field phenomenon.24
There are many areas for future work. Since detection of parafoveal capillaries was the goal of this article, there remains much work in image analysis for automation, enhanced segmentation, classification, and registration of vessels from motion contrast enhanced images. There are also important applications in clinical medicine based on FAZ quantification and capillary density, including the development of potential biomarkers for disease, measures of retinal health in disease progression, or endpoint measures for clinical trials.