Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x–z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 μm(3) with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 μm(3) in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-μm fluorescent beads.