New-generation of cryo-EM sample preparation device

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 570467104

Over the past years, cryogenic-electron microscopy (cryo-EM), including single-particle analysis and cryo-electron tomography, has revolutionized the field of structural biology. It allows the high-resolution 3D visualization of biological macromolecules in vitro and in situ, providing crucial insights into their molecular structures, mechanisms of action, function and regulation, as well as into cellular interaction and spatial and temporal cellular context. However, the success and the overall quality of cryo-EM data, heavily depend on the sample vitrification process, which aims to preserve the native conformation of the specimen while ensuring its imaging suitability. Yet, despite major technical advances in microscope hardware and image processing software that culminated in the structural biology ‘revolution’ experienced over the past years, the general sample preparation process in single-particle analysis has barely changed for several decades. In this conventional, non-automated workflow, several microliters of sample are manually applied on a grid, the excess volume removed by blotting paper and subsequently plunge-frozen into liquid ethane for further use. This workflow has several limitations, ultimately compromising success rate, data quality and applicability. It lacks full automation, offers poor reproducibility, and barely permits monitoring and fine-tuning of crucial parameters such as sample application strategies and freezing time, which are critical to obtaining consistent and reproducible optimal sample thickness while avoiding air-water inter-face artifacts and other deformation processes. Further, the blotting procedure itself is highly inefficient, discarding about 99% of the sample. Considering the ability of cryo-EM to reconstruct high-resolution 3D structures from only a few (hundred) thousand particles, which could be collected from only a few microliters of cells, highlights the importance and potential of new-generation cryo-EM sample preparation devices. The ability to, e.g., lower the require-ments of how much sample is needed by several magnitudes bears the potential to open up entirely new research lines, e.g., enabling structural determination of endogenously purified protein and protein complexes from scarce sources such as model system or patient material. Several strategies to optimize and automate sample preparation have been put forward over the past years, with only very few reaching a commercial level with convincing improvements. At the Heidelberg University, we want to stay at the forefront of cutting-edge science and apply this technology to our research. With the expertise and the appropriate research questions, this implementation bears the potential for enormous breakthroughs.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Neue Generation Kryo-EM-Probenvorbereitungsgerät
• Instrumentation Group: 5140 Hilfsgeräte und Zubehör für Elektronenmikroskope
• Applicant Institution: Ruprecht-Karls-Universität Heidelberg
• Leader: Professorin Dr. Cristina Paulino