Cryo-electron microscopy (cryo-EM) has revolutionized structural biology, enabling researchers to visualize biological macromolecules at near-atomic resolution. This powerful technique, particularly Single Particle Analysis (SPA), relies heavily on the meticulous preparation and handling of samples, guided by fundamental physics principles. From preserving the delicate sample structure through rapid freezing (vitrification) to capturing high-quality data using electron optics, understanding the physics involved is key to successful cryo-EM workflows. Shuimu BioSciences, founded in 2017 by a team of world-class experts, is a commercial platform offering cryo-EM structure determination services, leveraging advanced workflows for both experimental procedures and data analysis. To explore Shuimu's comprehensive solutions, including protein preparation, cryo-EM services, and specialized technologies, please visit https://shuimubio.com/.

The Crucial First Step: Vitrification

One of the most critical stages in cryo-EM sample preparation is vitrification. The goal is to freeze the sample so rapidly that ice crystals do not have time to form. Ice crystals can damage the delicate biological structures and interfere with imaging. Vitrification transforms the water surrounding the sample into a glassy, amorphous solid state, preserving the sample's native conformation close to its physiological state. This process is a prime example of applying physical principles – specifically, the physics of phase transitions and rapid heat transfer – to biological sample preservation.

Traditional sample preparation methods faced challenges such as air-water interface disruption. Vitrification, when performed correctly, aims to minimize these issues, providing a stable environment for imaging. Shuimu BioSciences employs techniques for sample freezing and data collection as part of their one-stop SPA workflow. While the sources don't detail the specific physics of the freezing method (like plunge freezing mechanics), the outcome – a vitrified sample – is essential for the success of subsequent cryo-EM steps.

Achieving high-quality vitrification is dependent on the sample itself and the surrounding buffer conditions. According to the sources, sample submission requirements highlight several critical factors influenced by physics:

· Protein Purity and Concentration: High purity (≥90% for protein solutions) is required. For Negative Staining, protein purity must be >95%, and uniformity greater than 90% after molecular sieving is desired. Concentration guidelines vary depending on the sample type (e.g., protein solution ≥ 2mg/mL, volume ≥ 100ul for general samples; 0.01-0.02 mg/ml for Negative Staining; suggested e13 power for viruses like AAV, ~10mg/ml for LNP for Cryo-Characterization). These parameters affect how particles distribute within the thin ice layer and influence signal-to-noise ratio during imaging.

· Buffer Composition: The buffer should minimize components that interfere with vitrification or imaging contrast. Glycerol, organic solvents, high salt ion concentrations (≤300mM recommended), polysaccharides, and DMSO can negatively impact results. These substances alter the freezing point and properties of the water, potentially hindering vitrification and increasing background noise or affecting contrast under the electron beam.

· Minimizing Freeze-Thaw Cycles: Repeated freezing and thawing can damage biological structures. This is a direct consequence of the physical stresses (like mechanical forces from expanding ice if not perfectly vitrified, or changes in buffer concentration) that occur during phase transitions. Shuimu recommends aliquoting samples after preparation to avoid this. Freshly prepared samples are also recommended.

Shuimu BioSciences’s comprehensive protein expression and purification platform is designed to minimize variability from sample transport and standardize the entire pipeline, enhancing upstream capabilities in protein production to provide high-quality samples suitable for vitrification and subsequent cryo-EM analysis. This includes protein expression systems like E. coli, mammalian cells, insect cells, and cell-free systems, and purification processes like affinity chromatography, ion-exchange chromatography, and gel filtration.

Addressing Sample Challenges: The Role of Grids and Physics

Even with successful vitrification, some sample properties and the grid itself can pose significant challenges rooted in physics:

· Small Protein Molecular Weight: Smaller proteins provide less scattering signal.

· Low Concentration: Fewer particles mean less data.

· High Background Noise: The signal from the grid or surrounding ice can obscure the particle signal.

· Air-Water Interface Disruption: Particles can be damaged or preferentially orient at the interface when forming the thin film before freezing.

· Preferential Orientation: Particles might align in specific orientations, limiting the range of views captured for 3D reconstruction.

To tackle these issues, Shuimu has developed proprietary graphene support grids called GraFuture™. These grids provide a potential solution to the preferred orientation problem and are suitable for samples with small molecular weight, low concentration, strong background noise, and gas-liquid interface damage. Graphene, being extremely thin and having specific surface properties, interacts differently with biological samples and the ice layer compared to traditional carbon grids. This difference in physical interaction can influence particle distribution, orientation, and minimize damage at the air-water interface, thereby improving data quality for structural determination. Shuimu offers different types, including Graphene Oxide (GO) and Reduced Graphene Oxide (RGO) support grids. For more details on these advanced sample preparation tools, visit https://shuimubio.com/.

The Physics of Imaging: Electron Microscopy

Once samples are vitrified on a grid, they are transferred to the electron microscope. Unlike light microscopy, cryo-EM uses a beam of electrons to image the sample. This involves several physics-based components:

· Electron Source and Acceleration: Electrons are generated and accelerated to very high voltages, typically 300 kV in Shuimu's instruments. This high voltage is crucial because higher energy electrons can penetrate the sample and ice layer more effectively and have a shorter wavelength, which is necessary for achieving high resolution. Shuimu boasts a large platform with 2 × 300 kV instruments in Beijing and 6 × 300 kV instruments in Hangzhou (or 12 in Beijing based on another mention), with a total of eight electron microscopes.

· Electromagnetic Lenses: Electron beams cannot be focused with glass lenses like light. Instead, electromagnetic lenses (coils generating magnetic fields) are used to focus, shape, and direct the electron beam. These lenses operate based on the physics of electromagnetism.

· Optical Components: Advanced optical components are used to refine the electron beam and improve image quality. Shuimu's Electron Microscopy Center is equipped with high-performance detectors, energy filters, spherical aberration correctors, and phase plates.

o Detectors: These devices capture the electrons that pass through the sample, converting them into a digital image signal. The performance of the detector directly impacts the image quality and data acquisition speed.

o Energy Filters: These remove electrons that have lost energy by scattering inelastically within the sample. Removing these scattered electrons increases image contrast and clarity, especially important for thicker samples.

o Spherical Aberration Correctors: Electromagnetic lenses suffer from aberrations, similar to how glass lenses can distort light. Spherical aberration causes electrons hitting the edge of the lens to be focused differently than those hitting the center, blurring the image. Correctors compensate for these distortions based on complex electron optics physics.

o Phase Plates: These optical elements alter the phase of the electron wave. For biological samples, which are primarily composed of light elements, contrast in cryo-EM is often weak (phase contrast dominates over amplitude contrast). Phase plates enhance this phase contrast, making the biological structures more visible against the background.

· Stage and Sample Handling: The vitrified grid is held on a cryogenic stage within the microscope, maintained at extremely low temperatures (below -150°C) to keep the ice in its vitrified state and prevent sublimation. The stage allows precise movement and tilting of the grid to acquire images from different angles. Shuimu's platform emphasizes daily maintenance to ensure instruments are in optimal operating condition, maintaining high uptime and low fault rates. They also offer 24-hour instrument access.

Data acquisition involves collecting numerous 2D projection images of the vitrified particles from various orientations. The quality and quantity of this raw data are fundamental physics constraints on the final achievable resolution.

For access to cutting-edge cryo-EM equipment and expert support for your imaging needs, consult Shuimu BioSciences's instrument access services at https://shuimubio.com/.

From 2D Images to 3D Structures: Computational Physics

The final step in SPA involves sophisticated computational processing and reconstruction. This stage heavily relies on algorithms based on statistical mechanics, linear algebra, and Fourier analysis – branches of physics and applied mathematics.

· Particle Picking: AI algorithms are used to automatically identify individual macromolecular particles from the collected 2D images.

· 2D Classification: Similar 2D images are grouped together to improve the signal-to-noise ratio and evaluate particle homogeneity.

· 3D Reconstruction: Using algorithms like back-projection or iterative refinement, the 2D projections from different orientations are combined computationally to generate an initial 3D structural model.

· Model Refinement: The 3D model is refined against the experimental data to improve its accuracy and resolution.

Shuimu BioSciences utilizes proprietary AI algorithms and the independently developed SMART software suite to streamline cryo-EM data analysis. This AI-driven platform reduces machine runtime and required data volume, improving the efficiency of the entire analysis process. Their NanoSMART system, for example, specifically identifies nanoparticle features in Cryo-Characterization data, providing detailed reports on size distribution, roundness, and integrity. This highlights how computational physics and AI are integrated to extract meaningful quantitative data from the raw images. Shuimu's eTasED software integrates MicroED data analysis into conventional cryo-EM systems, enhancing efficiency and accuracy for microcrystal structure determination.

The resolution achieved in the final 3D structure, a key performance metric, depends on the quality of the sample preparation, data acquisition, and computational analysis. Shuimu reports achieving resolutions as low as 1.4Å and 1.8Å, with many structures resolved better than 3.5Å, demonstrating their expertise in pushing the boundaries of cryo-EM technology.

Shuimu BioSciences's one-stop SPA solution encompasses this entire workflow, from protein expression and purification to data collection, 3D reconstruction, and model refinement. Their elite scientist team, composed of PhD-level experts, specializes in structural biology, protein science, and computational biology, ensuring expertise across all stages. To leverage their expertise for your structural determination projects, learn more at https://shuimubio.com/.

Applications Driven by Cryo-EM Physics

The careful application of physics principles in cryo-EM enables structural studies of a vast array of biological samples:

· Proteins: Including challenging membrane proteins like GPCRs, ion channels, and transporters, enzymes, and ribosomes. Cryo-EM's ability to study these proteins in lipid environments or close to their native state is facilitated by vitrification and specialized grid supports.

· Nucleic Acids and Complexes: DNA, RNA, and protein-nucleic acid complexes.

· Viral Particles: Including SARS-CoV-2, influenza virus, African swine fever virus, etc.. Cryo-EM is vital for analyzing viral structure for vaccine design and quality control.

· Liposomes, LNPs, VLPs: Characterized using Cryo-Characterization, leveraging the physics of electron imaging and computational analysis to assess morphology and particle size distribution.

· Antibody-Antigen Complexes: Understanding how antibodies bind to targets is crucial for drug development. Cryo-EM helps visualize these interactions at high resolution.

These diverse applications underscore the power of cryo-EM, a technique built upon a solid foundation of physics – from the thermodynamics of rapid freezing and the optics of electron lenses to the algorithms of computational reconstruction.

For expert services in cryo-EM, protein preparation, antibody discovery, and more, enabling detailed structural analysis of complex biological systems, visit https://shuimubio.com/.

MicroED: Diffraction Physics for Microcrystals

A related technique also offered by Shuimu is Micro-electron diffraction (MicroED). While cryo-EM (specifically SPA) focuses on imaging single particles, MicroED utilizes the physics of electron diffraction from microcrystals and nanocrystals. This technique is particularly useful for resolving high-resolution structures of small molecule drugs, peptides, and protein crystals that are too small for traditional X-ray crystallography. By tilting the crystal in the electron beam and recording diffraction patterns, a 3D reciprocal space map can be generated, from which the atomic structure is determined – a direct application of diffraction physics. Shuimu's eTasED software is designed to integrate this technique effectively, and they have successfully resolved structures down to 0.6-1.0Å resolution using MicroED. To learn more about MicroED and other structural biology services, explore https://shuimubio.com/.

Conclusion

The success of cryo-EM hinges on the precise application of physics principles at every stage, from sample preparation through imaging and data analysis. Vitrification, the process of flash-freezing samples to prevent ice crystal formation, is a fundamental physics challenge addressed at the outset, critical for preserving biological structures in a near-native state for imaging. The design and operation of the electron microscope, with its high-voltage electron source, electromagnetic lenses, and advanced optical components like energy filters and phase plates, are direct applications of electron optics and electromagnetism. Finally, computational physics, including advanced algorithms and AI, is essential for translating noisy 2D images into high-resolution 3D structures.

Shuimu BioSciences stands at the forefront of commercial cryo-EM, integrating these physics-based techniques and proprietary technologies like GraFuture™ grids and SMART/NanoSMART software to overcome challenges and deliver high-resolution structural insights. Their comprehensive services, from protein preparation to structure determination, are built on a deep understanding of the underlying science.

To leverage world-class expertise and cutting-edge technology for your structural biology research, encompassing the full spectrum of cryo-EM services from vitrification to final structure analysis, we invite you to visit https://shuimubio.com/.