A closer look at high-energy X-ray-induced bubble formation during soft tissue imaging

Improving the scalability of tissue imaging throughput with bright, coherent X-rays requires identifying and mitigating artifacts resulting from the interactions between X-rays and matter. At synchrotron sources, long-term imaging of soft tissues in solution can result in gas bubble formation or cavitation, which dramatically compromises image quality and integrity of the samples. By combining in-line phase-contrast cineradiography with operando gas chromatography, we were able to track the onset and evolution of high-energy X-ray-induced gas bubbles in ethanol-embedded soft tissue samples for tens of minutes (2 to 3 times the typical scan times). We demonstrate quantitatively that vacuum degassing of the sample during preparation can significantly delay bubble formation, offering up to a twofold improvement in dose tolerance, depending on the tissue type. However, once nucleated, bubble growth is faster in degassed than undegassed samples, indicating their distinct metastable states at bubble onset. Gas chromatography analysis shows increased solvent vaporization concurrent with bubble formation, yet the quantities of dissolved gases remain unchanged. Coupling features extracted from the radiographs with computational analysis of bubble characteristics, we uncover dose-controlled kinetics and nucleation site-specific growth. These hallmark signatures provide quantitative constraints on the driving mechanisms of bubble formation and growth. Overall, the observations highlight bubble formation as a critical, yet often overlooked hurdle in upscaling X-ray imaging for biological tissues and soft materials and we offer an empirical foundation for their understanding and imaging protocol optimization. More importantly, our approaches establish a top-down scheme to decipher the complex, multiscale radiation-matter interactions in these applications. Significance statement Better probing the X-ray radiation dose limit of bubble formation in biological tissue and developing mitigation methods is essential for improving imaging techniques involving X-ray, such as synchrotron X-ray tomography or crystallography. Here, we combined operando gas chromatography with in-line X-ray phase-contrast radiography on human lung and brain tissue to investigate bubble formation under high-energy X-ray irradiation. We demonstrate that vacuum degassing delays bubble nucleation up to a factor two, depending on the tissue type. Gas chromatography analysis showed increased solvent vaporization during bubble formation; however, the quantities of dissolved gases remained unchanged. Moreover, depending on the nucleation site, bubble growth can be geometrically constrained by sample microstructure, which influence its dynamics.