Label-free multimodal nonlinear optical imaging reveals key biomarkers of cellular radiation response

Abstract Ionizing radiation elicits complex and dynamic cellular responses that are not fully captured by conventional analytic approaches. Many existing assays typically rely on population-averaged measurements, require extensive and/or destructive sample preparation, or depend on exogenous contrast agents for single-cell analysis. Additionally, they often focus on isolated biomarkers rather than concurrent changes in key macromolecular constituents such as lipids and proteins. To address these limitations, we developed a label-free, multimodal nonlinear optical (NLO) imaging platform that integrates stimulated Raman scattering (SRS) microscopy at Raman shifts of 2850 cm⁻¹ and 2926 cm⁻¹ with two-photon excitation fluorescence (TPEF) microscopy targeting reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD). In foundational studies, we characterized the radiobiological effects of clinical X-ray doses (2–10 Gy) in human lung cancer A549 cells. Irradiated cells exhibited multiple quantifiable biomarkers that varied with radiation dose and post-irradiation time. These included changes in lipid and protein signals, differences in NAD(P)H and FAD intensities, higher lipid-to-protein ratios, enhanced optical redox ratio, enlarged cell and lipid droplet areas, and increased colocalization of lipid droplets with mitochondria. Collectively, these results establish NLO imaging as a sensitive and powerful modality for quantitatively assessing cellular responses to clinical doses of ionizing radiation.