Reverberation multiphoton microscopy

Fast, volumetric fluorescence imaging in scattering media has long been a huge challenge in microscopy. To date, multiphoton microscopy (MPM) is the only technique that enables deep penetration while preserving high resolution and contrast. But multiphoton microscopy is slow, and no effective technique has been reported yet to enable fast volumetric imaging over large depth ranges greater than a millimeter. We demonstrate such a technique. Our technique uses a reverberation cavity in the illumination path that splits laser pulses into an infinite series of beam foci, forming near-instantaneous axial scans while delivering the full illumination power to the sample. In this manner, we obtain simultaneous multiplane imaging from arbitrary depths (in principle, limited only by laser power) all the way to the sample surface.

• D. R. Beaulieu, I. G. Davison, K. Kilic, T. G. Bifano, J. Mertz, “Simultaneous multiplane imaging with reverberation  multiphoton microscopy”, Nat. Meth. 17, 283-286 (2020) link