In the past, microglia were broadly categorized into two “states”: “resting” and “activated”. In the healthy mature CNS, microglia have ramified morphologies with small soma and many thin processes, which were classified as in the “resting” state. However, any disturbance in the homeostasis of CNS, such as infection and injury, can trigger microglia to shorten processes and enlarge cell soma, resembling a more traditional macrophage-like “amoeboid” phenotype (Kettenmann et al., 2011). The phenotype shift is accompanied with changes in gene expression and functional behaviors, which were collectively summarized as “microglia activation” (Hanisch & Kettenmann, 2007; Kettenmann et al., 2011). Activated microglia can actively migrate to injury or infection sites following chemotactic gradients or increase local densities through proliferation (Hanisch & Kettenmann, 2007). Until recently, studies on microglia focused only on their roles as inflammatory cells in the contexts of CNS disturbances such as injuries and neurodegenerative diseases. However, recent studies showed that microglia are far more complex and the “resting” state microglia have been active in neuron network surveillance, protruding and retracting their processes constantly, contributing to neonatal synaptic pruning and adult synaptic plasticity (Harry, 2013; Schafer et al., 2012). In this thesis, we used multi-photon imaging along with other optical techniques to investigate microglia’s behaviors and functions in a variety of different scenarios.