This work presents the design and implementation of an enhanced version of a traditional two-photon (2P) microscope with the addition of high-speed axial scanning for live mammalian brain imaging. Our implementation utilizes a deformable mirror (DM) that can rapidly apply different defocus shapes to manipulate the laser beam divergence and consequently control the axial position of the beam focus in the sample. We provide a mathematical model describing the DM curvature, then experimentally characterize the radius of curvature as well as the Zernike terms of the DM surface for a given set of defocuses. A description of the optical setup of the 2P microscope is detailed. We conduct a thorough calibration of the system, determining the point spread function, the total scanning range, the axial step size, and the intensity curvature as a function of depth. Finally, the instrument is used for imaging different neurobiological samples, including fixed brain slices and in vivo mouse cerebral cortex.

Deformable mirror-based two-photon microscopy for axial mammalian brain imaging