Two-Photon Imaging of Microglia in the Mouse Cortex In Vivo

Microglia are the primary immune effector cells of the brain parenchyma. They are distributed throughout the brain at various densities. Two-photon fluorescence microscopy, together with expression of fluorescent proteins in microglia, has enabled study of these fascinating cells in vivo. Imaging studies have shown, for example, that microglia continually survey their cellular environment and immediately respond to injury. However, we still know very little about their roles in various parts of the developing and adult brain or their diverse effector functions in aging and different disease states. Experimental procedures have been developed for minimally invasive short- and long-term two-photon imaging of microglial cells in cortical regions of the intact mouse brain. This protocol describes two-photon imaging of microglia in the mouse cortex in vivo, using mice which have had a head plate implanted and have been prepared with either a thinned skull or optical window. Technical pitfalls, limitations, and alternative approaches are also discussed.