Noninvasive visualization and analysis of parafoveal capillaries in humans.

The retina is one of the most metabolically active tissues in the human body, and as such, is coupled with a unique system of blood vessels. The inner retinal microvasculature has been mapped out in high resolution using excised monkey retinas.1 Far from the fovea, the capillary network is multi-layered; closer to the fovea, the network thins down to two layers in the peri- to parafoveal region, and to one layer in the immediate parafoveal region. Finally, in the fovea, there are no vessels, forming the foveal avascular zone (FAZ). The average diameter of a normal FAZ as described in textbooks is around 400 to 500 μm. However, there is considerable individual variation.2 The FAZ has been well characterized and is of important clinical significance in a number of different diseases, including diabetes.3 There are two key challenges that must be addressed when performing optical imaging of the retinal microcirculation: low capillary contrast and aberrations in the eye. Contrast is typically improved using a contrast agent, such as in fluorescein angiography (FA). Although FA is considered to be a “gold standard” for studying retinal vessels, it does carry a small risk for adverse reactions.4 Due to these risks, FA is not performed on normal eyes, except under special circumstances. Moreover, FA may not be able to reliably identify the smallest capillaries.5 An alternate method to improve capillary contrast is to use video processing tools based on flow visualization.6 These tools included mean, variance, min, max, range, and transition images. The variance image has been previously applied as a method for increasing vessel contrast in microvessels before applying leukocyte tracking algorithms.7 However, the variance image alone fails to increase the contrast of vessels in our datasets. We will demonstrate that spatial and temporal information can be used to increase the local contrast for moving objects, and enable the visualization of capillaries. Aberrations in the eye, which may hinder the resolution of the smallest capillaries (i.e., those with diameter ∼5 μm), can be corrected using adaptive optics.8 Recently, an adaptive optics scanning laser ophthalmoscope (AOSLO) was used to quantify leukocyte speeds through parafoveal capillaries.9,10 In this article, we will combine video and image processing tools with AOSLO imaging to demonstrate an improved method that can detect even the smallest capillaries in the parafoveal region without the use of injected dyes.