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Multiphoton microscopy (MPM) has gained enormous popularity over the yearsfor its capacity to provide high resolution images from deep within scatteringsamples1. However, MPM is generally based on single-point laser-focus scanning,which is intrinsically slow. While imaging speeds as fast as video rate havebecome routine for 2D planar imaging, such speeds have so far been unattainablefor 3D volumetric imaging without severely compromising microscope performance.We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2Dplanar (single plane) imaging, with minimal compromise in performance.Specifically, multiple planes are acquired by near-instantaneous axial scanningwhile maintaining 3D micron-scale resolution. Our technique, calledreverberation MPM, is well adapted for large-scale imaging in scattering mediawith low repetition-rate lasers, and can be implemented with conventional MPMas a simple add-on.

Simultaneous multiplane imaging with reverberation multiphoton microscopy