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Structural Biology plays a crucial role in understanding the Chemistry of Life by providing detailed information about the three-dimensional structures of biological macromolecules such as proteins, DNA, RNA and complexes thereof. This knowledge allows researchers to understand how these molecules function and interact with each other, which forms the basis for a molecular understanding of disease and the development of targeted therapies. For decades, X-ray crystallography has been the dominant technique to determine these 3D structures. Only a decade ago, advances in technology and data processing resulted in a dramatic improvement of the resolution at which structures of biomolecular assemblies can be determined using another technique: cryo-electron microscopy (cryo-EM). This has been referred to as “the resolution revolution”. Since then, an ever increasing group of structural biologists are using cryo-EM. They employ a technique named Single Particle Analysis (SPA), in which thousands of individual macromolecules are imaged. These images are then computationally iteratively aligned and averaged to generate a three-dimensional reconstruction of the macromolecule. SPA works best if a very pure and concentrated macromolecule of interest can be captured in random orientations within a thin layer (10-50nm) of vitreous ice. Maastricht University has been the inventor of the machine that is found in most labs worldwide used for this: the VitroBot. We have been the inventor of succeeding technologies that allow for much better control of this process: the VitroJet. In here, we will develop a novel chemical way to expand our arsenal for preparing SPA samples of defined thickness. We will design, produce and test chemical spacers to allow for a controlled sample thickness. If successful, this will provide an easy, affordable solution for the ~1000 laboratories worldwide using SPA, and help them with their in vitro studies necessary for an improved molecular understanding of the Chemistry of Life.