3-photon fluorescence microscopy (3PFM) has emerged as a mean to image deep structures in biological tissues. 3PFM cannot be implemented on standard 2-photon fluorescence microscopes (2PFM), which use high repetition rate (~80MHz) lasers and nJ pulses. 3PFM requires lasers with high- energy pulses (μJ), at relatively low repetition rates (MHz and below). As a consequence of this low repetition rate, 3PFM images are obtained at low frame rates.

We will take advantage of our recently developed two-photon “Re-RAMP” microscope, in which acousto-optical deflectors (AOD) are synchronized with a low repetition rate laser to achieve ultrafast 2D and 3D scans and wavefront shaping. The Re-RAMP-3 microscope designed here will provide 3-photon random-access scanning in 3D with millisecond resolution and unprecedented depth penetration.

Its unique performances will be validated by experiments in neurosciences and developmental biology, facilitating its dissemination by Karthala System.