Sample Preparation Protocol for Laboratory Cryo-Soft X-Ray Microscopy for Studying Cellular Nanoparticle Uptake

Soft X-ray microscopy (SXM) is a powerful technique for high-resolution biomedical imaging, enabling the observation of bio-nano interactions in near-native conditions without the need for heavy metal staining and fluorescence labeling. A laboratory soft X-ray microscope (LSXM) was developed to bridge the resolution gap between light microscopy and electron microscopy in cellular imaging. However, LSXMs employ a lower-brightness X-ray source in comparison to those operated in synchrotron facilities, which can negatively affect the contrast of X-ray micrographs. Therefore, proper sample preparation is essential to achieve optimal imaging results. This paper details an LSXM sample preparation protocol for investigating cellular nanoparticle uptake. Samples are prepared using optimized parameters for both manual plunge-freezing and automated vitrification, ensuring the rapid transition of biological material into a solid state with controllable thickness in the 5-10 μm range, preserving cellular structures and enabling optimal X-ray transmission for cellular imaging. We demonstrate the effectiveness of this protocol in facilitating the observation of nanoparticle uptake in two different biological samples: murine macrophages and acanthamoeba. Controlling ice thickness improves X-ray transmission through the specimen, enhancing the contrast and image quality of SXM.