{
  "abstract": "Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.",
  "abstract_shown": true,
  "affiliations": [
    "TU Wien"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "name": "M. Pons Romero"
    },
    {
      "name": "B. Kalouskova"
    },
    {
      "name": "J. Kloimwieder"
    },
    {
      "name": "A. Linares"
    },
    {
      "name": "A. Majkova"
    },
    {
      "name": "L. Riedl"
    },
    {
      "name": "C. Kopittke"
    },
    {
      "name": "M. Bishara"
    },
    {
      "name": "M. Brameshuber"
    },
    {
      "affs": [
        0
      ],
      "name": "E. Sevcsik"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/fqb9fr525tc6s.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.24.754129",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754129v1.full",
  "id": "fqb9fr525tc6s",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754129v1",
  "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": "W7214534172",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754129v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-24",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/fqb9fr525tc6s.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:43Z",
      "native_id": "10.64898/2026.09.24.754129",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754129v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:biophysics",
      "subfield": "biophysics"
    }
  ],
  "title": "A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex",
  "title_text": "A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex",
  "type": "preprint",
  "updated_at": "2026-10-30T12:24:43Z",
  "url": "https://research.draconitic.com/paper/fqb9fr525tc6s",
  "venue": "bioRxiv",
  "versions": []
}