{
  "abstract": "Quantitative fluorescence lifetime imaging microscopy (FLIM) is limited by a lack of probes that combine single-residue specificity, minimal steric perturbation, visible-light excitation, and strong environment-dependent lifetime contrast. We introduce benzodiazole-based non-canonical amino acids (BD-ncAAs) as compact, genetically encoded sensors for site-specific FLIM. Using an engineered pyrrolysyl-tRNA synthetase pair, we incorporate three different BD-ncAAs into proteins in living cells. Site-specific incorporation of BD-ncAAs into a nanobody at different positions preserved native structure and antigen binding. The lead probe, cyano-substituted CBDS, exhibits visible-light excitation, large Stokes shifts, and pronounced environment-dependent fluorescence lifetimes spanning 1 to 24 ns. Under wash-free, live-cell FLIM conditions, CBDS quantitatively resolves localized structural microenvironments and transmembrane hydration gradients, while also distinguishing distinct states during protein secretion in eukaryotes. BD-ncAAs present genetically encoded, minimally perturbing reporters for tracking target proteins, detecting interactions with binding partners, and quantifying site-specific solvent accessibility in living cells by quantitative FLIM.",
  "abstract_shown": true,
  "affiliations": [
    "Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 11,
  "authors": [
    {
      "name": "H. Qianzhu"
    },
    {
      "name": "S. M. Butler"
    },
    {
      "name": "E. Habel"
    },
    {
      "name": "E. H. Abdelkader"
    },
    {
      "name": "N. Paul"
    },
    {
      "name": "R. Woolsey"
    },
    {
      "name": "X. Zhou"
    },
    {
      "name": "R. M. Martin"
    },
    {
      "name": "K. A. Jolliffe"
    },
    {
      "name": "G. Otting"
    },
    {
      "affs": [
        0
      ],
      "name": "T. Huber"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/13pyg61gt8rbq.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.13.758698",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1.full",
  "id": "13pyg61gt8rbq",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1",
  "language": null,
  "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",
      "biophysics"
    ],
    [
      "openalex",
      "1304|Advanced Fluorescence Microscopy Techniques"
    ]
  ],
  "openalex": "W7222865694",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-13",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/13pyg61gt8rbq.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:28:21Z",
      "native_id": "10.64898/2026.10.13.758698",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:biophysics",
      "subfield": "biophysics"
    }
  ],
  "title": "Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes",
  "title_text": "Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes",
  "type": "preprint",
  "updated_at": "2026-10-30T12:28:21Z",
  "url": "https://research.draconitic.com/paper/13pyg61gt8rbq",
  "venue": "bioRxiv",
  "versions": []
}