In our study, we did not regularly see the walls of the venules or capillaries, although occasionally we could measure the venule walls and detected hints of capillary walls. In addition the appearance of the vascular walls is more like a longitudinal cross section on our AOSLO images, whereas in reality we know the vessels are cylinders with the mural cells wrapping around the circumference of the vessels (this can be seen in the histology of
Fig. 6, right panel). In practice, we do see hints of these circumferential bands as shown in
Figures 2C3 and 6, left panel. However, we do not always see these though they must be present. Similarly, for the capillaries and venules we occasionally see hints of mural cells, but not reliably enough to quantify. The lack of visibility of venule and capillary pericytes could be due to their different structure. Capillary pericytes tend to be more rounded than on the precapillary arterioles
55 and on venules they are flattened against the endothelial cells. This raises the issue of what is limiting the detection in our imaging approach. The first issue is resolution. While we are essentially diffraction limited,
63 the structures we are imaging represent small variations in the index of refraction, but they are larger than the approximately 2-μm resolution limit for our imaging system. The second limitation is contrast, and issues here could arise either from the structure of the capillaries, the optical detection approach, or both. Because of the nature of the neurovascular unit the pericyte on capillaries is largely enveloped by the astrocyte endfeet,
55,56 which from our purposes of visualization likely decreases the contrast resulting in the smooth capillary walls we image. The optical limitations arise from the observation that we are using an approach that detects the vascular walls based on forward scatter that is in turn scattered back toward the pupil.
27 This imaging approach is closely related to phase contrast microscopy.
64 Like other methods sensitive to light scattering in retinal imaging,
24,65–68 the degree that the light beam interacts with the tissue is spatially limited. As the imaging beam passes over the vessels the first time, some of the light is diffracted at the vascular wall due to the change in refractive index. While the angle of diffraction is related to the spatial frequency of the wall structure, the amount of light diffracted is related to the total refractive index difference and the axial dimension of the structure within the focal volume. Imaging the vascular wall then becomes an issue of detecting this light that has been diffracted at an angle from the vessel and then scattered back toward the pupil.
27 Because we use a large displaced aperture, the AOSLO has a large depth of field. This means that we have a very large background signal, which arises from the out of focus elements of the retina and a relatively small signal from the vessel structure. We obtain contrast from the plane of focus because it is only here that the illumination beam is compact and a phase structure (the blood vessels) will interact with a significant proportion of the impinging light and therefore be diffracted; thus, whereas the amount of light diffracted is relatively small, the contribution of the nondiffracted light is large. The result is that the high frequency information from the larger vessel walls can be detected but the small amount from capillaries cannot be detected. The reason for not seeing the venule and capillary walls then becomes an issue of signal-to-noise ratio, and suggests that schemes to improve the detection of small signals on a large background, including real time background subtraction or suppression may enable further advances in vascular wall imaging.