High-speed multineuron calcium imaging using Nipkow-type confocal microscopy.

Conventional confocal and two-photon microscopy scan the field of view sequentially with single-point laser illumination. This raster-scanning method constrains video speeds to tens of frames per second, which are too slow to capture the temporal patterns of fast electrical events initiated by neurons. Nipkow-type spinning-disk confocal microscopy resolves this problem by the use of multiple laser beams. We describe experimental procedures for functional multineuron calcium imaging (fMCI) based on Nipkow-disk confocal microscopy, which enables us to monitor the activities of hundreds of neurons en masse at a cellular resolution at up to 2000 fps.