Assembly for semi-correlation analysis of samples using cryogenic scanning electron microscopy and cryogenic Raman micro-spectroscopy

Abstract Correlative imaging, integrating diverse observational methods, has become increasingly vital for comprehensive sample analysis by linking distinct datasets that are accessible only through individual techniques. This study combines cryogenic scanning electron microscopy (cryo-SEM) and cryogenic Raman micro-spectroscopy (cryo-Raman), which together offer extensive insight into sample analysis. Cryo-SEM effectively investigates the surface, structure, and morphology, while cryo-Raman excels in detecting and identifying chemical composition. To integrate these techniques, we developed a novel assembly compatible with commercial cryo-SEM sample holders. The assembly is designed for precise sample positioning and observation at cryogenic temperatures of liquid nitrogen. This setup, controlled by LabVIEW software, includes a Dewar vessel containing liquid nitrogen, a 3D-printed stage for mounting sample holders, and a piezoelectric stage for XY motion, with z axis adjustments connected to the Raman spectrometer table. A plexiglass cover minimizes contamination and preserves sample integrity during analysis. The assembly was validated by first analyzing polystyrene at cryogenic temperatures to ensure its functionality. Subsequent tests on Cupriavidus necator H16—bacteria capable of producing polyhydroxyalkanoates (PHAs), a type of biodegradable polyester—demonstrated the system’s efficiency. PHAs, which are of interest as a sustainable alternative to petrochemical plastics, can be produced from industrial waste streams. Our results show that the combination of cryo-SEM and cryo-Raman is suitable for studying these microorganisms and permits a deeper understanding of the properties of polymer granules in microbial cells. This integrated approach represents a significant advance in semi-correlative imaging, providing more efficient and detailed analyses for future research in microbial physiology production and related fields.