Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain
Abstract
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 x 230 um2 at 8.6 Hz at tissue depths of up to ~2 mm in mouse cortex and hippocampus.
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BibTeX
@misc{santra2026cellularresolution,
title = {{Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain}},
author = {Kalyan Santra and Francesca Logiacco and Agustina Frechou and Kevin Barber and Arash Bellafard and Jyun-you Liou and Alipasha Vaziri},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.16.759624},
url = {https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain
AU - Kalyan Santra
AU - Francesca Logiacco
AU - Agustina Frechou
AU - Kevin Barber
AU - Arash Bellafard
AU - Jyun-you Liou
AU - Alipasha Vaziri
PY - 2026
DA - 2026/10/16
PB - bioRxiv
DO - 10.64898/2026.10.16.759624
UR - https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1
AB - 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 x 230 um2 at 8.6 Hz at tissue depths of up to ~2 mm in mouse cortex and hippocampus.
SN - 2692-8205
ER -