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

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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.
Funding Information
  • Natural Sciences and Engineering Research Council of Canada (RGPIN-2022-04897)
  • Strategic Research and Development Program of Conexus Nuclear Inc.