MicroED: Revolutionizing Microcrystal Electron Diffraction

Determining the atomic structures of small, challenging crystals has long been a hurdle in structural biology and materials science. Traditional X-ray crystallography requires large, well-ordered crystals, which are often difficult to obtain. Electron-based techniques like Microcrystal Electron Diffraction (MicroED) offer an alternative by using much smaller crystals while achieving high-resolution structural insights.

Advancements in electron diffraction hardware and data processing have significantly improved the accuracy and efficiency of MicroED. Researchers can now solve structures that were previously inaccessible, opening new possibilities in drug discovery, chemistry, and material sciences.

Basic Principles Of Microcrystal Electron Diffraction

MicroED operates on the principle that electrons, due to their shorter wavelengths compared to X-rays, provide atomic-resolution structural data from much smaller crystals. A transmission electron microscope (TEM) directs a highly focused electron beam onto sub-micron-sized crystals, generating diffraction patterns that reveal atomic arrangements. Because electrons interact more strongly with matter than X-rays, they produce high-contrast diffraction patterns even from nanocrystals, making MicroED particularly useful for studying materials that are difficult to crystallize in larger forms.

A defining characteristic of MicroED is its continuous-rotation data collection, also known as “serial rotation electron diffraction” (SerialRED). Unlike traditional electron diffraction techniques that rely on static exposures, MicroED involves rotating the crystal at a constant rate while capturing diffraction frames in rapid succession. This approach minimizes radiation damage by distributing the electron dose across multiple orientations. The result is a more complete and higher-quality dataset, allowing for the determination of structures with resolutions often reaching 1.0 Å or better.

Radiation damage is a significant concern due to the high energy of electron beams, which can degrade sensitive biological and organic materials. To mitigate this, MicroED employs extremely low electron doses, typically 0.01–0.10 e⁻/Ų per frame, significantly lower than conventional electron crystallography methods. Cryogenic conditions, where samples are maintained at liquid nitrogen temperatures, further reduce structural alterations, ensuring that the final model accurately represents the native state of the molecule.

MicroED can analyze crystals in their native environments without extensive manipulation. Unlike X-ray crystallography, which requires large, well-ordered crystals, MicroED works with microcrystals that form naturally or as byproducts of failed crystallization trials. This capability has expanded the range of analyzable materials, including membrane proteins, small organic molecules, and pharmaceutical compounds. The technique has been particularly transformative in drug discovery, where rapid structure determination of small-molecule drugs and their polymorphs informs formulation strategies and intellectual property considerations.

Specimen And Grid Preparation

Preparing high-quality specimens for MicroED begins with selecting microcrystals that are sufficiently small and well-ordered for electron diffraction. Unlike X-ray crystallography, which demands large, single crystals, MicroED requires crystals ranging from 100 nm to a few micrometers. These must have minimal defects and high crystallinity to generate interpretable diffraction patterns. Sample preparation starts by suspending the microcrystals in a volatile buffer or solvent that preserves their structure while ensuring uniform dispersion. The choice of buffer is critical, as high-salt or viscous solutions can interfere with electron transmission and produce background noise.

Once a stable suspension is achieved, the microcrystals are applied to a TEM grid, typically copper or gold-based with a carbon or graphene oxide support film. Ultrathin carbon films are often preferred due to their minimal electron scattering properties, which enhance diffraction quality. A small volume of the crystal suspension is deposited onto the grid, followed by blotting away excess liquid to create a thin, uniform layer. This step is crucial, as excessive liquid can lead to crystal aggregation or uneven distribution, compromising data quality.

Vitrification rapidly freezes specimens, locking them in a near-native state. Plunge freezing in liquid ethane at cryogenic temperatures prevents damaging ice crystal formation. Maintaining samples at liquid nitrogen temperatures throughout the experiment minimizes beam-induced damage and preserves structural fidelity. Cryo-TEM grids are then transferred to the microscope under controlled conditions to prevent contamination or devitrification.

Grid screening helps identify well-dispersed, high-quality crystals suitable for diffraction. Low-dose imaging techniques locate individual crystals while minimizing radiation exposure. Automated software assists in selecting the best candidates based on crystal size, orientation, and diffraction potential. Poorly oriented or aggregated crystals are avoided. Optimizing ice thickness during vitrification is also crucial, as overly thick ice can obscure diffraction signals, while excessively thin layers may cause dehydration or mechanical stress.

Data Acquisition Techniques

Capturing high-quality diffraction data in MicroED requires precise control over electron beam parameters, crystal orientation, and detector sensitivity. The process begins with selecting an optimal acceleration voltage, typically between 200 and 300 kV, to balance penetration depth and diffraction quality. Higher voltages improve electron transmission through thicker crystals, but excessive energy increases radiation damage, requiring a careful trade-off. The beam is tuned to a parallel or nearly parallel configuration to ensure uniform illumination, minimizing distortions in the diffraction pattern.

Once the electron beam is optimized, continuous-rotation data collection begins. The crystal rotates at a controlled speed—often between 0.2° and 1.0° per second—while diffraction frames are recorded in rapid succession using a highly sensitive direct electron detector or hybrid pixel detector. Direct electron detectors improve data quality by reducing noise and enhancing signal-to-noise ratios, allowing for the detection of weak reflections. This method, known as continuous rotation electron diffraction (cRED), captures reflections from multiple orientations with minimal gaps, providing a more complete dataset.

Dose fractionation mitigates radiation damage by distributing the total electron dose across multiple frames, typically 0.01–0.10 e⁻/Ų per frame. This preserves structural integrity while maintaining high-resolution diffraction signals. Low-dose imaging techniques locate and center crystals before data collection, ensuring only the most suitable specimens are analyzed. Automated data collection software streamlines this process, enabling high-throughput screening of multiple crystals within a single session.

Processing And Interpretation Of Data

Once diffraction frames are collected, raw data must be processed to extract meaningful structural information. The first step involves correcting distortions introduced by the electron optics and detector, ensuring that diffraction spots accurately represent the crystal’s atomic lattice. This includes addressing beam-induced motion, which can blur diffraction spots, and correcting for detector-specific artifacts. Software packages such as DIALS and XDS have been adapted for MicroED, allowing for precise indexing and integration of diffraction peaks. Because MicroED produces continuous-rotation data, algorithms must track reflections across successive frames, capturing the full three-dimensional diffraction volume of the crystal.

After integration, scaling and merging procedures refine the dataset by compensating for variations in crystal thickness, beam intensity fluctuations, and partial reflections. These adjustments enhance internal consistency and improve signal-to-noise ratios. Unlike traditional electron crystallography, where phase information is often a limiting factor, MicroED benefits from direct methods and molecular replacement strategies commonly used in X-ray crystallography. By leveraging known structural templates or ab initio phasing, researchers can determine atomic positions with remarkable accuracy, even for complex molecules.

Types Of Crystals Used In MicroED

MicroED analyzes extremely small crystals unsuitable for conventional structural techniques. These include biological macromolecules, small organic compounds, and inorganic minerals, each presenting unique challenges and advantages.

Biological Macromolecules
Proteins and peptides that resist crystallization into large, well-ordered structures have been a major focus of MicroED. This includes membrane proteins, which typically form microcrystals due to their amphipathic nature, and amyloid fibrils, which assemble into nanocrystalline domains. MicroED allows researchers to determine structures of these biomolecules at near-atomic resolution, even when only sub-micron crystals are available. A notable example is the structure of the toxic core of α-synuclein, a protein linked to Parkinson’s disease. Unlike X-ray crystallography, which requires extensive optimization of crystallization conditions, MicroED works with naturally occurring microcrystals, making it especially useful for studying biologically relevant conformations.

Small Organic Molecules and Pharmaceuticals
MicroED has proven invaluable in pharmaceutical research, where resolving the structures of drug molecules and their polymorphs is essential for formulation and intellectual property protection. Many pharmaceutical compounds crystallize in microcrystalline forms that are difficult to analyze through traditional methods. MicroED enables rapid structure determination, often requiring only nanograms of material. This technique has been used to characterize drug polymorphs, which can exhibit different solubility and stability properties, directly impacting bioavailability and efficacy.

Inorganic and Hybrid Crystals
Beyond biological and pharmaceutical applications, MicroED has been instrumental in solving structures of inorganic materials, including zeolites and metal-organic frameworks (MOFs). These materials often form as fine powders, making them challenging for single-crystal X-ray diffraction. MicroED overcomes this limitation by utilizing nanocrystals to determine atomic arrangements with precision. The structural analysis of zeolites, widely used in catalysis and gas separation, has benefited from MicroED’s ability to resolve framework connectivity and pore structures. Similarly, MOFs, which have applications in gas storage and drug delivery, can be characterized more efficiently through MicroED, accelerating the discovery and optimization of new materials.