Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

The uniformity of the protein patterns, their shape, and the contrast between the fluorescence signal of the pattern and the background, critically modulate the quantitative accuracy of the microarray-derived data. While significant research focused of the identification of the factors that impact the protein microarray patterns, these studies usually have focused on the optimization of one set of these factors, e.g., how the spot uniformity is affected by different additives, or by different surfaces. However, the complex interaction between proteins, carrier fluids, surfaces, and patterning methodologies used would suggest a systematic and more comprehensive study that considers all these parameters, as well as their inter-relationship. The present work compared the patterning of two fluorescently-tagged proteins, i.e., IgG, BSA, on surfaces with different hydrophobicity and chemistry, and printed by inkjet, pin, and microcontact printing (µCP). The quantification of the spot size regularity, its morphology, the signal intensity and its distribution within spots were used to assess the quality of a specific printing method, on a specific surface, with a specific solute of the printed protein. It was found that the optimal uniformity for both droplet-based methods depend on surface chemistry, with glass slides modified with 3-Glycidoxypropyl-dimethoxymethyl silane (GPS) and 3-(Aminopropyl)-triethoxy silane (APTES) exhibiting the greatest uniformity, while uniformity of the µCP patterns was relatively independent of the surface chemistry. For the inkjet and pin printing, the largest fluorescence signal and contrast with the background was found on APTES modified glass slides, whereas for the µCP the fluorescence signal increased with increasing hydrophilicity.