Microglia, the resident innate immune cells of the brain, are known to perform key roles such as synaptic pruning, apoptotic debris removal, and pathogen defense in the central nervous system. Microglial mutations are directly linked to many neurodevelopmental (e.g., schizophrenia) and neurodegenerative (e.g., Alzheimer's disease) disorders, indicating the diagnostic and therapeutic potential of microglia for treating these conditions. Currently, we lack robust molecular tools to specifically image and manipulate microglia in vivo, which presents a major hurdle in our understanding of the brain-wide functions of these cells during the early onset of brain diseases. Here, we describe a molecular technology for imaging and manipulation of homeostatic microglia in live organisms (e.g., in zebrafish and mice) by covalently targeting the purinergic receptor, P2RY12. Using this technology, we imaged microglia-pathogen interactions in the larval zebrafish brain and revealed various morphological states of microglia in the adult mouse brain. We further expanded the microglia labelling approach to single-microglia tracking and microglial surfaceome mapping using photoactivatable fluorophores and photoproximity labelling, respectively. We anticipate the use of this universal tool for studying microglial biology across species to reveal the dynamics and polarization of resting microglia into a reactive state found in many neurodegenerative diseases. ### Competing Interest Statement The authors have declared no competing interest.