Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call \"honeycomb gold discs\", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.

Palytoxin (PTX) is a potent neurotoxin found in marine animals that can cause serious symptoms such as muscle contractions, haemolysis of red blood cells and potassium leakage. Despite years of research, very little is known about the mechanism of PTX. However, recent advances in the field of cryoEM, specifically the use of microcrystal electron diffraction (MicroED), have allowed us to determine the structure of PTX. It was discovered that PTX folds into a hairpin motif and is able to bind to the extracellular gate of Na,K-ATPase, which is responsible for maintaining the electrochemical gradient across the plasma membrane. These findings, along with molecular docking simulations, have provided important insights into the mechanism of PTX and can potentially aid in the development of molecular agents for treating cases of PTX exposure.

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

The preparation of transmission electron microscopy (TEM) samples from powders is quite difficult and challenging. For powders with particles in the 1-5 μm size range, it is especially difficult to select an adequate sample preparation technique. Epoxy is commonly used to bind powder, but drawbacks, such as differential milling originating from unequal milling rates between the epoxy and powder, remain. We propose a new, simple method for preparing TEM samples. This method is especially useful for powders with particles in the 1-5 μm size range that are vulnerable to oxidation. The method uses solder as an embedding agent together with focused ion beam (FIB) milling. The powder was embedded in low-temperature solder using a conventional hot-mounting instrument. Subsequently, FIB was used to fabricate thin TEM samples via the lift-out technique. The solder proved to be more effective than epoxy in producing thin TEM samples with large areas. The problem of differential milling was mitigated, and the solder binder was more stable than epoxy under an electron beam. This methodology can be applied for preparing TEM samples from various powders that are either vulnerable to oxidation or composed of high atomic number elements.

An advanced software-controlled focused ion beam (FIB) patterning process for the fabrication of combined atomic force-scanning electrochemical microscopy (AFM-SECM) probes is reported. FIB milling is a standard process in scanning probe microscopy (SPM) for specialized SPM probe fabrication. For AFM-SECM, milling of bifunctional probes usually requires several milling steps. Milling such complex multi-layer/multi-material structures using a single milling routine leads to significantly reduced fabrication times and costs. Based on an advanced patterning routine, a semi-automated FIB milling routine for fabricating combined AFM-SECM probes with high reproducibility is presented with future potential for processing at a wafer level. The fabricated bifunctional probes were electrochemically characterized using cyclic voltammetry, and their performance for AFM-SECM imaging experiments was tested. Different insulation materials (Parylene-C and SixNy) have been evaluated with respect to facilitating the overall milling process, the influence on the electrochemical behavior and the long-term stability of the obtained probes. Furthermore, the influence of material composition and layer sequence to the overall shape and properties of the combined probes were evaluated.