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bioRxiv · 10.64898/2026.01.12.699031

Establishing label-free quantitative X-ray dose-response profiling by Holo-Tomographic Flow Cytometry

Pirone, D.·de Vita, C.·Mottareale, R.·Giugliano, G.·Giordano, G.·Grilli, S.·Bianco, V.·Miccio, L.·Memmolo, P.·Durante, M.·Pugliese, M.·Ferraro, P.
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Abstract

Flow cytometry (FC) offers multiparametric analysis capabilities that can quantify cellular damage after exposure to cytotoxic agents. Here, we present a comprehensive study establishing a label-free quantitative X-ray dose-response profiling using a novel FC modality based on 3D Quantitative Phase Imaging, termed Holo-Tomographic Flow Cytometry (HTFC). This approach enables fully label-free 3D refractive index (RI) measurements, allowing detailed and quantitative characterization of the biophysical properties and morphology of living cells exposed to X-rays. By analyzing datasets of 3D RI tomograms from cells irradiated at graded doses, we identify intracellular biophysical markers that define a robust X-ray dose-response curve. Validation against standard clonogenic survival assays on three model cancer cell lines reveal a high correlation (>90%). HTFC not only eliminates labeling and operator bias but also markedly reduces experimental time from 1-2 weeks to 24 hours, offering a fully automated and objective readout. While clonogenic survival remains the benchmark for radiosensitivity assessment, our findings establish HTFC as a powerful label-free platform for fast assessment of radiation damage. This technology paves the way for predictive biosensors that can capture patient-specific responses, thereby supporting the transition from conventional, uniform radiotherapy protocols to personalized treatment strategies.

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Pirone, D., de Vita, C., Mottareale, R., Giugliano, G., Giordano, G., Grilli, S., Bianco, V., Miccio, L., Memmolo, P., Durante, M., Pugliese, M., Ferraro, P.. 2026-01-12. Establishing label-free quantitative X-ray dose-response profiling by Holo-Tomographic Flow Cytometry. https://doi.org/10.64898/2026.01.12.699031

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