Video-rate large-scale imaging with multi-Z confocal microscopy

Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this challenge, we report an augmented variant of confocal microscopy that uses a series of reflecting pinholes axially distributed in the detection space, such that each pinhole probes a different depth within the sample. We thus obtain simultaneous multiplane imaging without the need for axial scanning. Our microscope technique is versatile and configured here to provide two-color fluorescence imaging with a field of view larger than a millimeter at video rate. Its general applicability is demonstrated with neuronal imaging of both Caenorhabditis elegans and mouse brains in vivo.