Microglia primarily reside in the inner and outer plexiform layers of the adult retina, but an additional population can be visualized in the RNFL and at the ILM. With the advancement of SD-OCT imaging, macrophage-like cells can be visualized on the human ILM [6]. Without immunohistochemistry analysis, though, it is difficult to determine the specific cell types. In normal retinas, those cells might be resident macrophages or hyalocytes.

By performing eight hourly scans, we were able to demonstrate cell translocation over time in a healthy subject and to visualize the morphology of these cells.

In their resting state, macrophages visualized within 3 µm of the ILM of the healthy retina have a quiescent phenotype characterized by small somata and ramified filopodia-like processes (Fig. 2B) that actively survey their environment. The time-lapse en-face OCT of our healthy subject has demonstrated the macrophages’ dynamic behavior with continuous movement of their processes (Supplementary video). They appear in constant motion, extending and retracting to survey their environment.

Dynamic SD-OCT imaging in our control subject found that a minority of cells visualized in the posterior hyaloid (within 100 µm from the ILM) are highly mobile and less ramified, suggesting that this subpopulation could be hyalocytes. Their number is low in normal physiological conditions. Hyalocytes were first described in the 1840s and are considered resident macrophages of the cortical vitreous based on traditional macrophage marker expression [7]. After retinal damage, hyalocytes can change their morphology, density, and cell signaling.

In conclusion, by employing dynamic SD-OCT, we can visualize microglial cells on the surface of the retina, and by layering the scans, we can distinguish two different cell populations: one larger and moving with amoeboid movement, consistent with residents’ macrophages and one smaller and more dynamic, consistent with hyalocytes. Since, microglia dysregulation promotes the progression of some retinal vasculature diseases (such as diabetic retinopathy), our identification of macrophages as a component of the retinal surface population is an important finding. Furthermore, compared to retinal macrophages, human hyalocytes express pro-angiogenic and pro-chemotactic transcriptional profiles, suggesting their potential role in the inflammatory component of diabetic retinopathy and retinal vein occlusions. As such, if properly visualized in vivo, hyalocytes could become inflammation biomarker. Identification of inflammatory biomarkers is an important step toward personalized medicine.

Finally, our identification of microglia near the vitreoretinal interface suggests that human imaging of macrophages could be a biomarker for microglia-driven diseases like multiple sclerosis.