Imaging the dynamics of individual processes of microglia in the living retina in vivo.

Microglia are an essential population of resident immune cells in the central nervous system (CNS) and retina. These microscopic cells possess sub-cellular processes that make them challenging to image due to limited resolution and contrast. The baseline behavior of microglial processes in the living retina has been poorly characterized, and yet are essential to understanding how these cells respond under conditions of health, development, stress and disease. Here we use in vivo adaptive optics scanning light ophthalmoscopy combined with time-lapse imaging and quantification of process motility, to reveal the detailed behavior of microglial cells in a population of healthy mice. We find microglial processes to be dynamic at all branch-levels, from primary to end-protrusions. Cell-processes remodel at average speeds of 0.6 ± 0.4 µm/min with growth and deletion bursts of 0-7.6 µm/min. Longitudinal imaging in the same mice showed cell-somas to remain stable over seconds to minutes, but show migration over days to months. In addition to characterizing in vivo process motility and Sholl analysis using a microglial reporter mouse, we also demonstrate that microglia can be imaged without fluorescent labels at all. Phase-contrast imaging using safe levels of near-infrared light successfully imaged microglia soma and process remodeling with micron-level detail noninvasively, confirmed by simultaneous imaging of fluorescent microglial cells in transgenic mice. This label-free approach provides a new opportunity to investigate CNS immune system noninvasively without requiring transgenic or antibody labeling which could have off-target effects of changing normal microglial behavior. Additionally, CNS microglia study can now be conducted without the need for cranial window surgery which have the potential to change their behavior due to local or systemic inflammation.