Automated segmentation and analysis of retinal microglia within ImageJ

Microglia are immune cells of the central nervous system capable of distinct phenotypic changes and migration in response to injury. These changes most notably include the retraction of fine dendritic structures and adoption of a globular, phagocytic morphology. Due to their characteristic responses, microglia frequently act as histological indicators of injury progression. While algorithms seeking to automate microglia counts and morphological analysis are becoming increasingly popular, few exist that are adequate for use within the retina and manual analysis remains prevalent. To address this, we propose a novel segmentation routine, implemented within FIJI-ImageJ, to perform automated segmentation and cell counting of retinal microglia. We show that our routine could perform cell counts with accuracy similar to manual observers using the I307N Rho model. Tracking cell position relative to retinal vasculature, we observed population migration towards the photoreceptor layer beginning 12 h post light damage. Using feature selection with Chi² and principal component analysis, we resolved cells along a morphological gradient, demonstrating that extracted features were sufficiently descriptive to capture subtle morphological changes within cell populations in I307N Rho and Balb/c TLR2^-/- retinal degeneration models. Taken together, we introduce a novel automated routine capable of efficient image processing and segmentation. Using data retrieved following segmentation, we perform morphological analysis simultaneously on whole populations of cells, rather than individually. Our algorithm was built entirely with open-source software, for use on retinal microglia.