{
  "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.",
  "abstract_shown": true,
  "affiliations": [],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 7,
  "authors": [
    {
      "name": "Kalyan Santra"
    },
    {
      "name": "Francesca Logiacco"
    },
    {
      "name": "Agustina Frechou"
    },
    {
      "name": "Kevin Barber"
    },
    {
      "name": "Arash Bellafard"
    },
    {
      "name": "Jyun-you Liou"
    },
    {
      "name": "Alipasha Vaziri",
      "orcid": "0000-0001-8346-7551"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/2ct3y7q92zzr5.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.16.759624",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1.full",
  "id": "2ct3y7q92zzr5",
  "issns": [
    "2692-8205"
  ],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1",
  "language": "en",
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "neuroscience"
    ],
    [
      "heading",
      "Article"
    ],
    [
      "openalex",
      "1304|Advanced Fluorescence Microscopy Techniques"
    ]
  ],
  "openalex": "W7222978755",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1.full.pdf",
  "pmcid": "PMC13738460",
  "pmid": "42995871",
  "published": "2026-10-16",
  "publisher": "Cold Spring Harbor Laboratory Preprints",
  "ris": "https://research.draconitic.com/paper/2ct3y7q92zzr5.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:28:29Z",
      "native_id": "10.64898/2026.10.16.759624",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.16.759624v1"
    },
    {
      "first_seen": "2026-11-09T12:23:26Z",
      "native_id": "PMC13738460",
      "source": "pmc",
      "url": "https://doi.org/10.64898/2026.10.16.759624"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:neuroscience",
      "subfield": "neuroscience"
    }
  ],
  "title": "Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain",
  "title_text": "Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain",
  "type": "preprint",
  "updated_at": "2026-11-09T12:23:26Z",
  "url": "https://research.draconitic.com/paper/2ct3y7q92zzr5",
  "venue": "bioRxiv",
  "versions": []
}