Quantification of microglial dynamic motility in vivo

Motility is a fundamental property of microglial processes. Their dynamic movement is highly sensitive to alterations in the surrounding microenvironment and serves as an early indicator of pathological conditions in the central nervous system (CNS). Thus, comprehensive and accurate quantification of microglial process dynamics is critically needed. To address this, we developed a novel approach based on 3D alignment of original two-photon time-lapse images to correct misalignments caused by microglial migration or animal motion. This alignment facilitates subsequent colocalization analysis, allowing quantification of complete microglial structural dynamics at single-pixel resolution. Compared to conventional methods, our technique offers enhanced comprehensiveness and precision, enabling simultaneous measurement of process extension or retraction, microglial loss or regeneration, and clear visualization of temporal structural changes. Finally, we quantified the dynamic motility of microglia in abilateral common carotid artery occlusion/reperfusion (BCCAO/R) model using this method. We anticipate that this method will significantly improve the quantitative accuracy of dynamic motility assessments for microglia and other cell types in vivo.