Establishing label-free quantitative X-ray dose-response profiling by holo-tomographic flow cytometry

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 to 2 weeks to 24 h, 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.