Quasi-simultaneous multiplane calcium imaging of neuronal circuits.

Two-photon excitation fluorescence microscopy is widely used to study the activity of neuronal circuits. However, the fast imaging is typically constrained to a single lateral plane for a standard microscope design. Given that cortical neuronal networks in a mouse brain are complex three-dimensional structures organised in six histologically defined layers which extend over many hundreds of micrometres, there is a strong demand for microscope systems that can record neuronal signalling in volumes. Henceforth, we developed a quasi-simultaneous multiplane imaging technique combining an acousto-optic deflector and static remote focusing to provide fast imaging of neurons from different axial positions inside the cortical layers without the need for mechanical disturbance of either the objective lens or the specimen. The hardware and the software are easily adaptable to existing two-photon microscopes. Here, we demonstrated that our imaging method can record, at high speed and high image contrast, the calcium dynamics of neurons in two different imaging planes separated axially with the in-focus and the refocused planes 120 µm and 250 µm below the brain surface respectively.