Label-Free Optical Imaging Uncovers Key Biomarkers of Cellular Radiation Response
Abstract
Purpose
To apply label-free multimodal nonlinear optical (NLO) imaging including stimulated Raman scattering (SRS) and two-photon excitation fluorescence (TPEF) microscopy for multiplex single-shot identification of metabolic biomarkers of radiobiological response to clinical X-ray doses in human lung cancer cells.
Methods
Human lung adenocarcinoma epithelial A549 cells were irradiated with 100 kVp X-rays at 2, 4, 6, 8, and 10 Gy with parallel unirradiated controls. Dishes were then left in an incubator for 24, 48, and 72 hours prior to imaging the live cells. A femtosecond laser system with fixed (1040 nm, 200 fs) and variable (680–1300 nm, 130 fs) outputs was used to acquire SRS images at 2850 cm-1 (lipids) and 2926 cm-1 (proteins) and TPEF images of endogenous reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) fluorescence. These datasets were used to evaluate candidate biomarkers: protein and lipid content per cell, fluorescence output per cell, lipid/protein (L/P) ratio, optical redox ratio (ORR), cell area, and lipid droplet (LD) area. Changes in biomarkers were evaluated as a function of dose and post-irradiation time using a Wilcoxon rank sum test.
Results
Lipid content, protein content, and NAD(P)H and FAD fluorescence output per cell all increased significantly compared to control cells (p < 0.01) with dose and time. L/P ratio increased with dose and remained stable in time. Irradiated cells had stable ORR values over time, while ORR decreased with time for cells dosed at 4 Gy and below. Cell area and LD area both significantly increased compared to control cells (p < 0.01) with dose and time.
Conclusion
This work establishes that NLO imaging is a sensitive modality capable of uncovering mechanistic signatures and heterogeneity of single cell radiation response. Identifying these biomarkers has the potential to improve therapeutic outcomes and enhance understanding of radiation’s biological effects.