Fast z-focus controlling and multiplexing strategies for multiplane two-photon imaging of neural dynamics

Monitoring neural activity and associating neural dynamics with the anatomical connectome are required to understand how the brain works. Neural dynamics are measured by electrophysiology and optical imaging. Since the discovery of the two-photon excitation phenomenon, significant progress has been made in deep imaging for capturing neural activity from numerous neurons in vivo. The development of two-photon microscopy is aimed to image neural activity from a large and deep region with high spatial (x, y, and z) and temporal (t) resolutions at a high signal-to-noise ratio. Imaging deep regions along the optical axis (z-axis) is particularly challenging because heterogeneous biological tissues scatter and absorb light. Recent advances in the light focus modulation technology at high speeds in three dimensions (x, y, and z) have allowed multiplane two-photon imaging. z-Focus control by varifocal optical systems, such as ferroelectric liquid lenses, gradient refractive index lenses, and adaptive optical element systems, and multiplexing by time- and wavelength-division strategies have allowed to rapidly observe specimens at different focal depths. Herein, we overview the recent advances in multiplane functional imaging systems that enable four-dimensional (x, y, z, and t) analysis of neural dynamics, with a special emphasis on z-scanning mechanisms and multiplexing strategies.