Cellular-resolution three-photon ultra-deep imaging (TrUDI) of neuronal population activity at 2-mm depth in the mammalian brain

Extending the depth reach of multiphoton microscopy for imaging neuronal population activity in the scattering mammalian brain is an 'exponentially hard' problem. Three-photon (3p) microscopy has enabled imaging of neuroactivity at depths up to ~1.2 mm in the mouse brain; however, the fundamental depth limit of 3p imaging, as set by the depth at which out-of-focus background fluorescence exceeds the in-focus fluorescence signal, is well beyond 2 mm. Reaching this fundamental depth limit in 3p imaging has been hampered by insufficient in-focus fluorescence signal. Here we demonstrate a three-photon ultra-deep imaging (TrUDI) system that approaches this fundamental depth limit via systematic and principled optimization of the in-focus fluorescence signal. TrUDI matches the parameters of the excitation laser, the optical system, and the acquisition to linear and nonlinear limits for safe laser exposure while satisfying the spatiotemporal requirements of single-cell resolution calcium (Ca²⁺) imaging. TrUDI achieves a ~250-fold increase in detected fluorescence photon yield at the same laser power as in previous approaches thereby enabling in vivo cellular-resolution recordings of neuronal population activity within fields of view of ~230 × 230 µm² at 8.6 Hz at tissue depths of up to ~2 mm in mouse cortex and hippocampus.