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 multi-photon 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 the 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 arising from suboptimal optical design and acquisition conditions. 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 optimally matches the parameters of the excitation laser, the optical system, and the acquisition to linear and non-linear limits for safe laser exposure. At the same time, TrUDI remains compatible with the spatial and temporal requirements for single-cell resolution calcium imaging, and its parameters are tuned to the photo-physics of 3p excitation and the scattering properties of the mouse brain tissue. Our approach results in a ∼250-fold increase in detected fluorescence photons at the same laser power as in previous approaches. We use TrUDI to demonstrate in vivo cellular-resolution recordings of neuronal population activity within fields of view of ∼230 × 230 µm ² at 8.6 Hz at tissue depths of up to ∼2 mm in mouse cortex and hippocampus.