A simple pressure-assisted method for MicroED specimen preparation

MicroED enables macromolecular structure determination from nano- to micron-sized crystals but sample preparation is a critical bottleneck. Traditional cryo-EM blotting workflows often remove microcrystals and are ineffective for viscous mother liquors (e.g., high-PEG buffers or lipidic cubic phase), limiting MicroED’s applicability. The study aims to develop and validate a simple, universal specimen preparation method that preserves crystals, works across viscosities, and allows control of ice thickness. The authors introduce Preassis, a pressure-assisted approach that removes excess liquid through the EM grid, hypothesizing it will improve crystal retention, accommodate viscous buffers, and enable tunable ice thickness for high-quality MicroED data.

Prior work established MicroED for rapid structure determination with continuous-rotation data collection and standard crystallographic processing. However, preparing thin, uniform vitrified ice with intact sub-micrometer crystals is challenging, especially with viscous precipitants (e.g., PEGs) or LCP used for membrane proteins. Conventional pipetting–blotting–plunging often removes crystals and fails to adequately remove viscous liquids. Alternatives include manual backside blotting, direct crystallization on grids, and cryo-FIB milling, but a simple, broadly applicable method remains lacking. Crystal-size control typically involves tuning crystallization, mechanical fragmentation, or cryo-FIB, each with limitations.

Concept and setup: Preassis removes excess liquid through the EM grid using suction/pressure. In the simplest setup, a glow-discharged holey carbon EM grid rests on filter paper at the mouth of a Buchner flask connected to a vacuum source. A 3 µl droplet of crystal suspension is applied; suction underneath pulls liquid through the grid. The grid is then manually lifted with tweezers and plunge-frozen in liquid ethane. Ice thickness is tuned by pressure level, grid carbon hole size, and application time. In the presented setup, pressure is adjusted via pumping speed (approximately linear between 20–80% speed), and ice thickness may vary across the grid due to non-uniform contact with the filter paper.

Comparative study: Preassis was compared to Vitrobot double-side blotting using tetragonal lysozyme microcrystals (non-viscous, high concentration) for crystal density, data quality, and ice thickness, with multiple grids per condition. Humidity effects on ice thickness were assessed using ZSM-5 microcrystals in 40% PEG 400.

Specimen preparation parameters (Preassis):

• Lysozyme (orthorhombic): Quantifoil R 1.2/1.3; pressures 17.2 and 27.7 mbar; ~5 s from application to plunge; ~20 °C, ~35% or 80% RH.
• Lysozyme (tetragonal): Quantifoil R 1.2/1.3; pressures 0 and 37.2 mbar; ~5 s; ~20 °C; 80% RH also tested.
• R2lox (44% PEG 400): Quantifoil R 3.5/1; 30.7 mbar; 5 s; ~20 °C; 35% or 80% RH.
• Human dynamin I BSE-GTPase (30% PEG 4000): Quantifoil R 3.5/1; 181 mbar; 10 s.
• Viscosity study (inorganic proxy): ZSM-5 crystals in PEG 6000 (15%, 25%, 35% w/v) and PEG 400 (40% v/v); grids R 1.2/1.3, R 2/1, R 3.5/1; pressures 0 or 181 mbar; ~10 s (PEG 6000) or ~5 s (PEG 400) at ~20 °C, ~35% or 80% RH.

Vitrobot controls:

• R2lox: R 3.5/1; 4 °C; 100% RH; 10 s; 2 layers of paper per pad; blot force 16.
• Tetragonal lysozyme: R 1.2/1.3; 20 °C; 80% RH; single blot; 5 s; blot force 5.
• ZSM-5 in 40% PEG 400: R 3.5/1; 20 °C; RH 35%, 80%, or 100%; single blot; 5 s; blot force 5.

Imaging and data collection:

• JEOL JEM-2100LAB (200 kV), Gatan 914 cryo-holder; Gatan Orius camera for imaging; SAED and MicroED on Timepix detector; typical settings: spot size 3, camera length 80–100 cm, 1–2 s/frame; continuous rotation at 0.45° s−1; dose ~0.10 e− Å−2 s−1; Instamatic software.
• Thermo Fisher Themis Z (300 kV) with monochromator and Gatan OneView (for Fig. 2 and Supplementary Tables datasets): atlas imaging via Instamatic; MicroED with spot size 11, Mono-50, camera length 2.3 m, dose 0.03 e− Å−2 s−1, rotation 0.57° s−1, 2 s/frame; processing by XDS.

Evaluation metrics: crystal density on grids (TEM imaging), ice thickness/visibility (grid transparency, ice rings in ED), humidity impact on ice, and MicroED data statistics (resolution where I/σ ≥ 1, mean I/σ, Rmeas, CC1/2).

• Crystal retention: With 500× diluted tetragonal lysozyme, Preassis grids contained thousands of microcrystals whereas Vitrobot grids were nearly empty. Vitrobot required ≥250-fold higher crystal concentration (2× dilution of original) to achieve similar coverage. Preassis preserves roughly two orders of magnitude more crystals than Vitrobot.
• Data quality equivalence: From top 3 datasets per grid, Vitrobot vs Preassis averaged: resolution (I/σ ≥ 1) 2.67(11) Å vs 2.57(8) Å; I/σ 5.0(5) vs 5.1(7); Rmeas 0.314(29) vs 0.320(28); CC1/2 0.966(7) vs 0.963(14)—indicating comparable MicroED data quality given suitable ice thickness and crystal density.
• Viscous buffers and humidity robustness: For ZSM-5 in 40% PEG 400, Vitrobot’s grid transparency decreased ~4-fold at 80–100% RH compared to 35% RH, yielding few usable squares. Preassis produced mostly transparent squares at both 35% and 80% RH, demonstrating more efficient removal of viscous liquid and reduced humidity sensitivity.
• Case study—R2lox (44% PEG 400): Vitrobot (even with extreme blotting: dual papers, force 16, 10 s) yielded thick ice, few crystals, and poor diffraction (to ~9.0 Å). Preassis (30.7 mbar, R 3.5/1) produced thin ice with increased crystal density; diffraction improved to 3.0 Å, enabling sufficient high-quality MicroED data from a single grid and enabling the first novel MicroED protein structure of R2lox.
• Tunable ice thickness: Ice can be optimized by pressure and grid hole size. For orthorhombic lysozyme, increasing pressure from 17.2 to 27.7 mbar on R 1.2/1.3 thinned ice and removed ice rings. Using larger holes (R 3.5/1) at the same pressure also thinned ice and improved ED without ice rings. Excessive pressure combined with large holes led to signs of dehydration and reduced resolution.
• Viscosity guidelines: For non-viscous samples, usable grids can be obtained even at 0 mbar with appropriate hole sizes and timing; for PEG 6000 ≤25%, large-hole grids (R 3.5/1) can work without pressure but ice is often thicker; applying ~181 mbar improves specimens. For very viscous suspensions (e.g., 35% PEG 6000), high pressure is necessary; for extremely viscous LCPs, Preassis may need to be combined with viscosity-reducing strategies (detergents, oils, lipase).
• Practical selection: Optimal conditions can involve either higher pressure with smaller holes or lower pressure with larger holes. Hole size should be slightly smaller than or comparable to the largest crystal dimension to prevent crystal loss while maximizing usable hole area.

The study addresses the key bottleneck in MicroED—specimen preparation—by introducing a pressure-assisted method that preserves crystals, reduces dependence on blotting efficiency, and allows fine control of ice thickness. By pulling liquid through the grid, Preassis mitigates crystal loss inherent to standard blotting and more effectively handles viscous mother liquors, while also being less sensitive to environmental humidity. Comparable MicroED data quality to Vitrobot under optimal conditions and superior performance under challenging (viscous, low-concentration) conditions demonstrate its broad applicability. The ability to tune ice via pressure and grid hole size provides flexibility to accommodate different crystal sizes and morphologies, improving throughput for MicroED, including serial crystallography approaches where many crystals are advantageous. The improved R2lox diffraction and resultant structure determination exemplify the method’s impact on enabling previously stalled projects.

Preassis is a simple, low-cost, and widely accessible pressure-assisted approach for MicroED specimen preparation that enhances crystal retention, accommodates a wide range of buffer viscosities, and enables tunable ice thickness via pressure and grid selection. It yields MicroED data quality comparable to conventional methods under favorable conditions and surpasses them for viscous or low-density samples, as demonstrated by improved R2lox diffraction. The authors provide practical guidelines for selecting pressure, hole size, and timing across sample viscosities and crystal morphologies. Future work should focus on automating Preassis, integrating environmental control to improve reproducibility and throughput, optimizing growth of intrinsically small microcrystals, and developing improved screening methods for submicron crystals.

• Ice thickness can vary across the grid due to non-uniform grid–filter paper contact in the simple setup, potentially impacting reproducibility.
• Excessive pressure combined with large hole sizes can cause partial dehydration of crystals and reduced diffraction quality, indicating a need for careful parameter optimization.
• Extremely viscous samples (e.g., lipidic cubic phase) may still require additional treatments (detergents, oils, lipase) or complementary methods to reduce viscosity.
• Method performance is sample- and morphology-dependent; pressure needs to be tuned to crystal size/shape to avoid crystal loss or dehydration.
• MicroED still requires sufficiently small crystals (preferably ≤ ~500 nm in one dimension); when crystals are too large, additional steps (segmentation or cryo-FIB) may be necessary, which are not optimal solutions.