Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this issue, we developed a variant of confocal microscopy that provides simultaneous multiplane imaging over large field of view and at video rate. Our apparatus, called multi-Z confocal microscopy, differs from a conventional confocal microscope in both its illumination and detection parts. First, axially elongated illumination is achieved by under filling the back aperture of the microscope objective. The resulting low NA provides an axial extent of the illumination of the order of 100 µm. The light from the sample is then collected by the same objective, now taking advantage of the full NA which ensures high collection efficiency, and send to the detection unit. The latter is comprised by four reflecting pinholes axially distributed in the image plane such that they are conjugated to different depths within the sample. Each detection channel spans a probe volume at a different depth and volumetric imaging is obtained by simply combining the four channels.
In our current configuration, each imaging plane covers a field of view of 1.2 mm and the distance between two planes is equal to 25 µm. In other words, we image 1200x1200x100 µm3 at 30Hz. We first demonstrated the applicability of our technique by imaging entire C. elegans in vivo with a cellular resolution. Secondly we applied our technique to image multiple layers of neurons in mouse brain. We were able to record the activity of 550 neurons, with 100-150 neurons present in each imaging plane.