In vivo two-photon calcium imaging using multicell bolus loading

This protocol describes an approach, multicell bolus loading (MCBL), for in vivo two-photon Ca(2+) imaging of large neuronal circuits with the resolution of individual cells. The procedure was developed for in vivo imaging of the cortex, but it can be adapted easily for imaging other brain regions, including the cerebellum, olfactory bulb, or even individual spinal cord neurons. The staining technique can also be applied in brain slices of any developmental stage, from embryonic to adult. If combined with a miniature head-mounted two-photon microscope, it may allow in vivo two-photon imaging in freely moving animals.