This chapter overviews the use of acetoxymethyl (AM) ester-based multi cell bolus loading (MCBL) technique for in vivo Ca2+ imaging of neural circuits. This technique provides anatomically targeted, rapid and non-invasive staining of both neurones and glia with small molecule Ca2+ indicators thus allowing real-time imaging of brain activity. We describe the protocols for staining cells in newborn, juvenile, adult and aged tissue; discuss critical steps and possible pitfalls and introduce the multicolor imaging approach for functional characterization of specific cell types. We show, furthermore, that the use of MCBL can be extended to label other elements within the brain tissue as, for example, amyloid plaques in a mouse model of Alzheimer’s disease (AD). Finally, we illustrate the use of MCBL for two-photon calcium imaging of neurones and glia in the aged mouse cortex as well as the mouse cortex in an animal model of AD.