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Understanding Radiation Damage in Cryo-Electron Microscopy

Radiation damage in cryo-electron microscopy (cryo-EM) poses a significant challenge as it affects both the sample and the quality of an image. This ultimately impinges the resolution of derived structural information and therefore is important to understand how radiation damage occurs in cryo-electron microscopy and develop strategies to reduce it.

The Basics of Radiation Damage

Radiation damage during EM imaging occurs when an unstained biological specimen is exposed to high-energy electrons. Energy transferred from incident electrons to the specimen lead to a range of physical and chemical changes. The primary forms of radiation damage include:

Radiolysis: The energy from electrons can break chemical bonds, particularly in sensitive areas such as protein side chains, leading to molecular fragmentation and the generation of free radicals.
Mass Loss: As a consequence of radiolysis, volatile components may be released from the specimen, leading to mass loss and changes in the overall composition.
Charging of the sample: due to secondary electron emission of the sample (much like in a scanning electron microscope), a net positive charge is achieved which can ultimately lead to a catastrophic breakdown of the specimen.

These types of damage collectively degrade the structural integrity of the specimen, leading to artifacts in the final cryo-EM images. The resulting data can exhibit blurring, loss of contrast, and other distortions that complicate the interpretation of the molecular structures.

Factors Influencing Radiation Damage

Several factors influence the extent of radiation damage in cryo-EM:

Electron Dose: The degree of radiation damage is entirely proportional to the total amount of electron exposure, measured in electrons per square angstrom (e-/Å²). A higher dose increases the likelihood of atomic displacements and chemical bond breakages.
Specimen Composition: Different materials and biomolecules vary in their susceptibility to radiation damage. For example, organic molecules with high hydrogen content are more prone to damage than heavier elements.
Temperature: Cooling the specimen to cryogenic temperatures (~100 K or even down to 4K) reduces thermal vibrations and can mitigate some radiation effects. Also, fragments arising from bond breakage can remain trapped in a matrix of ice at very low specimen temperatures, thus seemingly avoiding a loss of resolution. However, even at these low temperatures, radiation damage is not entirely preventable.
The accelerating voltage: Lower voltages, i.e. 80 or even 30 kV, will result in more damage as the interaction volume of electrons increases at lower voltages. Thus higher voltages like 200 or 300 kV are commonly used for obtaining high-resolution structures using single particle analysis approaches. It should be noted, however, that the type of damage has some correlation with the voltage. That is, at voltages above 400 kV straight displacement by the electron will result in so-called knock-on damage.

Cryo-EM and the Electron Dose

Cryo-EM is used to image biomolecular complexes that are unstained and often without fixatives. Biological structures are obtained in their vitreous state by flash-freezing samples in liquid ethane kept close to its freezing point by liquid nitrogen. Statistically well-defined images that allow one to obtain the structure are impossible to get from such samples as they would instantaneously evaporate under the intense electron beam. Thus, images are obtained at a much lower dose but since these images are very noisy extensive image processing techniques are required. To establish what dose can be used researchers have in the past resorted to using two-dimensional crystals and monitored the fading of Bragg reflections in diffraction patterns as a function of electron dose. These have resulted in guidelines for resolving 3Å details with a total dose of 10-30 e-/Å². However, if one were to study structures at moderate resolutions, say 1 nm, a much higher dose can be used.

Advances in Mitigating Radiation Damage

Several technological and methodological advances have been developed to minimize radiation damage in cryo-EM:

Energy Filtering: Energy filters can be used to remove inelastically scattered electrons that contribute to noise and degrade image quality. By filtering out these electrons, the resulting images have improved contrast.
Phase plates: These devices, placed in the electron beam path, will affect imaging in much the same way as they do in light microscopes. By altering the phase of one part of the electrons a different contrast mechanism is utilized resulting in a much higher signal to noise ratio of the images compared to traditional, defocus-dependent imaging.
Cryo-EM Automation: Automated cryo-EM systems can optimize data acquisition parameters in real-time, adjusting the electron dose and focus to minimize damage while maintaining image quality. These systems also enable high-throughput data collection, reducing the time each specimen spends under the electron beam.
Direct Electron Detectors: The introduction of direct electron detectors has significantly improved the efficiency of cryo-EM. These detectors have higher sensitivity and faster readout speeds, allowing for lower electron doses while maintaining image resolution.

The concept of dose fractionation has emerged as a key strategy in addressing this challenge. Instead of exposing the specimen to a single high dose, the total dose is divided into multiple smaller doses, and images are captured sequentially. These images are later combined computationally to produce a final image that is of higher resolution due to the fact that specimen motions arising from radiation damage events are negated by aligning the sequence of images.

The Future of Cryo-Electron Microscopy

As cryo-EM technology continues to evolve, the ability to mitigate radiation damage will further enhance the technique’s capabilities. Researchers are exploring new approaches such as phase plate technology, which enhances contrast without increasing the electron dose, and advanced image processing algorithms that can better compensate for radiation-induced artifacts.

Learn more about the JEOL CRYO ARM™

As cryo-EM continues to evolve, managing radiation damage remains crucial for achieving accurate, high-resolution results. The CRYO ARM™ from JEOL offers a sophisticated solution to this challenge. The JEOL CRYO ARM™ series, including the CRYO ARM™ 300 II, minimizes electron dose and enhances image clarity, preserving sample integrity for cryo-electron microscopy. Features like the Zero Fringe System ensure uniform illumination while reducing beam damage, and a high-precision stage allows efficient sample handling. Combined with advanced automation via JADAS 4 software, the CRYO ARM™ empowers researchers to explore complex biological structures with speed, precision, and confidence, ensuring high-quality data acquisition.

References and further reading:

Lindsay A. Baker, John L. Rubinstein. Chapter Fifteen - Radiation Damage in Electron Cryomicroscopy. Editor(s): Grant J. Jensen. Methods in Enzymology. Academic Press. Volume 481. 2010. Pages 371-388. ISSN 0076-6879. ISBN 9780123749062. https://doi.org/10.1016/S0076-6879(10)81015-8.

A Beginner's Guide to Cryo-Electron Microscopy (Cryo-EM)

Structural biology has undergone a seismic shift with the rise of cryogenic electron microscopy (cryo-EM). Once a niche technique, cryo-EM is now at the forefront of molecular and cellular imaging, providing researchers with the ability to visualize biological structures at near-atomic resolution. But what exactly is cryo-EM, and why has it become such a pivotal tool in modern science? This guide will take you through the basics of cryo-EM, its key methodologies, and its transformative impact to the fields of structural biology.

What is Cryo-EM?

Cryo-electron microscopy, or cryo-EM, is a form of electron microscopy where samples are studied at cryogenic temperatures (below -150°C). Unlike traditional methods that may require extensive sample preparation, including staining or crystallization, cryo-EM allows researchers to observe biological specimens in their near-native state, frozen in a thin layer of vitreous ice. This technique has revolutionized our ability to study complex biological molecules, viruses, and cellular components in unprecedented detail.

Transmission Electron Microscopes, the backbone of cryo-EM, use electrons instead of light. The interaction between the electrons and the specimen yields high resolution images, which can be further analyzed to extract structural information.

The Importance of Cryo-EM in Structural Biology

Cryo-EM's rise to prominence can be traced back to its ability to fill a crucial gap in structural biology. While X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have long been the go-to methods for determining molecular structures, they come with limitations. X-ray crystallography requires well-ordered crystals, which are often difficult or impossible to obtain for large or flexible molecules. NMR, while powerful, is best suited for smaller proteins but not for large macromolecular complexes.

Cryo-EM, on the other hand, is routinely used to solve large and complex structures that are challenging for other techniques. It has proven particularly valuable in resolving the structures of macromolecular assemblies, membrane proteins, and even entire viruses. The ability to visualize these structures in their near-native environments has provided insights that were previously out of reach.

A Brief History of Cryo-EM

The story of cryo-EM began in 1933 when Max Knoll and Ernst Ruska developed the first transmission electron microscope. This breakthrough was a major milestone in microscopy, enabling scientists to observe particles much smaller than what was possible with optical microscopes. It laid the groundwork for future developments in electron microscopy, including the cryogenic techniques that would come decades later.

In the 1970s, scientists faced a significant challenge: how to minimize radiation damage to biological samples during electron microscopy. The solution emerged from cryogenic techniques, which examined samples at extremely low temperatures. This approach, validated using two-dimensional or thin three-dimensional crystals showed a substantial reduction in radiation damage out to high resolution at temperatures close to liquid nitrogen or even liquid helium. In the same decade, Joachim Frank pioneered the development of image processing techniques that would become essential to cryo-EM. Frank’s work focused on transforming the often noisy and blurry 2D images captured by electron microscopes into clear, interpretable 3D structures. This ability to extract meaningful structural information from cryo-EM data was a game-changer for the field, allowing scientists to visualize complex biological molecules in unprecedented detail.

A stumbling block to studying biological systems in their native state is the presence of water and the requirement for the electron microscope to be kept at high vacuum. Parsons was able to circumvent this using a wet cell, or environmental chamber, in the microscope employing a set of membranes to keep the protein in water whilst avoiding the protein-destroying boil-off that the vacuum would cause. Currently, a large body of work exists using this very principle to study in-situ events using special purpose specimen holders. Yet, the large amount of water in a wet cell would result in substantial loss of signal and the radiation damage caused at room temperature meant that this technique would not result in high-resolution structures. A major leap forward occurred in 1981 when Jacques Dubochet and Alasdair McDowall introduced the method of rapid freezing, or vitrification. Thus, a specimen carrier with a small amount of water was quenched in liquid ethane kept close to its melting point using liquid nitrogen. The rapid cool down, occurring at a rate of roughly 105K/sec, would ensure crystalline water would not form and the sample would be suspended in a thin layer of vitreous ice. This innovation was crucial in protecting specimens from dehydration and damage from ice crystals enabling researchers to observe them in a native state.

Cryo-EM's full potential became evident in 1990 when Richard Henderson and his colleagues determined the first atomic resolution structure of a biomacromolecule—bacteriorhodopsin—using cryo-EM applied to two-dimensional crystals. The entire amino acid sequence could be folded into the three-dimensional map of the protein. This achievement demonstrated that cryo-EM could produce atomic-level details of biological structures, opening new possibilities for research in structural biology.

A transformative leap in cryo-EM was made in 2012-2013 with the introduction of direct electron detectors. These new detectors captured images using a movie mode, meaning that the primary obstacle in cryo-EM, beam-induced motion, could be largely corrected by aligning the individual frames in the movie. This breakthrough led to significantly improved image quality and resolution, resulting in what is colloquially known as the "resolution-revolution" in cryo-EM. Along with improved automation and reliability of the electron microscopes, this development marked the technique's transition into a highly reliable and widely adopted method for determining the structures of complex biomolecules.

The importance of cryo-EM was globally recognized in 2017 when the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson. Their pioneering contributions to the development of cryo-EM were acknowledged as a breakthrough that had fundamentally changed the field of structural biology.

How Cryo-EM Works

The process of cryo-EM can be broken down into several key steps:

Sample Preparation: The first and most critical step in cryo-EM is the preparation of the sample. A purified sample is applied to a grid, and excess liquid is blotted away, leaving a thin film of the sample. This grid is then rapidly plunged into liquid ethane, freezing the sample at a rate that prevents ice crystal formation and preserves the sample in a vitreous state. Many variations exist on this topic ranging from the type of carrier film used, such as carbon, graphene (derivatized or not) or gold foils for instance, to the method of applying the sample, applying either a droplet or dispensing the sample onto a grid using a stylus or an inkjet technique to name a few. Carrier film with holes at regular intervals are routinely used at this stage.
Data Collection: Once the sample is vitrified, it is loaded into a transmission electron microscope operating at cryogenic temperatures. Often, many grids of the same sample are loaded as freezing can still produce variation from sample to sample meaning subtly different imaging conditions are needed. Specific holes as identified from low-magnification images are targeted at higher magnification after which multiple images are acquired with a direct detector using beam shift plus image shift techniques in order to minimize the time required for the stage to settle. Typically, thousands of images and millions of particle images are needed for a high-resolution structure, even more so if the sample exhibits conformational flexibility.
Image Processing: The captured images are subjected to a process called motion correction, where individual frames of the images are aligned to account for any movement of the sample during imaging. Next, the effects of defocus on each image is analyzed in a process called CTF estimation. Particles are then selected and extracted from the micrographs as 2D images, and sorted based on similarity in a process called 2D classification. Finally, 2D classes are chosen and used to reconstruct a final 3D model, which can be further refined to improve resolution and used to fit in amino acid chains and build a structure.

Applications and Future Directions

As a powerful and versatile tool in structural biology, cryo-EM provides researchers with unparalleled insights into the molecular architecture of biological systems. Its broad range of applications and the promising future directions for this technology continue to drive advancements in various fields.

Structure Determination of Biomolecules

Cryo-EM has become the method of choice for determining the structures of large and complex biomolecules at near-atomic resolution. This technique excels in visualizing macromolecular assemblies that are difficult to study using traditional methods like X-ray crystallography or NMR spectroscopy. Notable examples of structures elucidated by cryo-EM include protein complexes, viruses, ribosomes, membrane proteins, and spliceosomes.

New methods are being developed to extend cryo-EM’s reach to more challenging and diverse samples:

Membrane Proteins and Lipid-Protein Complexes: Techniques are being refined to better study these difficult targets, which are vital for understanding cellular processes and drug interactions.
Intrinsically Disordered Proteins: Cryo-EM is evolving to capture the structures of these flexible and dynamic proteins, which play critical roles in cell signaling and regulation.
Large Macromolecular Assemblies: As cryo-EM technology improves, it will become increasingly feasible to study very large complexes, shedding light on their intricate workings.

Drug Discovery and Development

The pharmaceutical industry has increasingly adopted cryo-EM as a key tool in drug discovery and development. Its ability to rapidly provide high-resolution structures of drug targets enables the design of more effective therapeutics. The atomic structures of complexes provided by cryo-EM also open the door to rational drug design using powerful docking tools of ligands, cofactors or effector molecules into active sites. Applications in this area include structure-based drug design, fragment-based drug discovery, PROTACs development, and antibody drug development.

The integration of cryo-EM into drug discovery is expected to grow, with several promising applications:

High-Throughput Screening: Cryo-EM could be used for the rapid screening of drug candidates, accelerating the early stages of drug development.
Studying Drug-Target Interactions: The ability to visualize these interactions at atomic resolution will provide insights that are crucial for designing more effective drugs.
Investigating Drug Resistance: Cryo-EM will help researchers understand the structural basis of drug resistance, guiding the development of next-generation therapeutics.

Cellular and Molecular Biology

Cryo-EM’s ability to capture the detailed architecture of cellular components has made it an invaluable tool in cellular and molecular biology. Key applications include studying protein-protein interactions, examining conformational changes, and investigating cellular organelles.

Advancing cryo-EM techniques to study biomolecules within their native cellular environments will open new frontiers in structural biology:

Cryo-Electron Tomography: This approach will allow for the 3D visualization of intact cells, providing insights into the spatial organization and interactions of cellular components.
Correlative Light and Electron Microscopy: Combining cryo-EM with light microscopy will enable the study of dynamic processes within cells, bridging the gap between molecular structures and cellular functions.

Closing Thoughts

Cryo-EM has firmly established itself as a powerhouse in structural biology. Its ability to provide detailed images of biological structures at near-atomic resolution has opened new frontiers in our understanding of molecular and cellular processes. As the technology continues to evolve, cryo-EM is set to play an even more central role in future scientific discoveries. Whether you're a seasoned researcher or a curious beginner, the world of cryo-EM offers endless opportunities for exploration and discovery.

References & Further Reading:

Savva C. A beginner's guide to cryogenic electron microscopy. Biochem (Lond). 2019;41 (2):46–52. doi:10.1042/BIO04102046.
Cheng Y. Single-particle cryo-EM-How did it get here and where will it go. Science. 2018;361(6405):876-880. doi:10.1126/science.aat4346.
Benjin X, Ling L. Developments, applications, and prospects of cryo-electron microscopy. Protein Sci. 2020;29(4):872-882. doi:10.1002/pro.3805.
Du YM, Gao YZ, Jia XD, et al. Applications and prospects of cryo-EM in drug discovery. Military Medical Research. 2023;10(1):10. doi:10.1186/s40779-023-00446-y.

What is Single-Particle Cryo-EM?

Single-particle cryo-electron microscopy (cryo-EM) has emerged as one of the most powerful techniques in structural biology. By providing near-atomic resolution of biomolecules in their native states, this revolutionary technology has enabled researchers to uncover the structure of proteins, enzymes, and other macromolecular complexes that are difficult or impossible to study with traditional methods, such as x-ray crystallography or NMR. JEOL’s advancements in cryo-EM systems, like the CRYO ARM™ series, have played a pivotal role in expanding the scope of this technique for structural biology and drug discovery applications.

Key Features of Single-Particle Cryo-EM

The cryo-EM process involves three key stages to generate high-quality structural insights:

Sample Preparation and Flash-Freezing: Biological samples purified by biochemical techniques are flash-frozen in a thin layer of vitreous ice to preserve their native state. This rapid freezing prevents the formation of damaging ice crystals, ensuring the structural integrity of the sample is maintained in a near-physiological form.
Electron Microscopy Imaging: The samples are imaged after freezing in a transmission electron microscope under cryogenic conditions, where images of the individual particles are captured as thousands of 2D projections at varying orientations. These images are crucial for generating a particle dataset, which is subjected to various processing algorithms.
Computational Reconstruction: The particle images are computationally aligned, averaged, and processed to reconstruct a high-resolution 3D structure. By combining these 2D projections, advanced software determines the most probable spatial arrangement of the particles, enabling the creation of near-atomic-resolution models, all without the need for crystallization.

This workflow provides precise structural insights into complex biological macromolecules, often revealing details unattainable by traditional methods like X-ray crystallography.

Advantages of Single-Particle Cryo-EM

Cryo-EM is celebrated for several distinct advantages over other structural biology techniques, such as X-ray crystallography:

No Need for Crystallization: One of the most significant benefits of cryo-EM is its ability to study proteins that are difficult or impossible to crystallize. This opens new avenues for research on large protein complexes and membrane proteins. Additionally, macromolecular complexes can be studied by cryo-EM that are simply too large for NMR.
Native-State Imaging: Unlike other techniques that may require staining or fixation, cryo-EM allows researchers to image proteins and macromolecular complexes in their natural, hydrated state without chemical alterations.
Multi-State Resolution: Cryo-EM can resolve multiple conformational states of a protein from the same sample. This ability to capture different functional states offers valuable insights into protein dynamics and mechanisms.
High Resolution: With modern cryo-EM technologies, atomic or near-atomic resolution is achieved routinely, allowing detailed visualization of the atomic structure of macromolecules.

Applications in Structural Biology

Cryo-EM has dramatically impacted structural biology, enabling scientists to solve complex biological questions that were once beyond reach. Here are some key applications:

Large Protein Complexes: Cryo-EM is particularly useful for determining the structures of large, multi-subunit protein complexes that are too large or flexible for other techniques. For example, complexes like the ribosome and proteasome have been elucidated using cryo-EM.
Membrane Proteins: Membrane proteins, crucial for various biological processes, are notoriously difficult to crystallize. Cryo-EM provides a means to study their structures, aiding in the understanding of cell signaling and transport mechanisms.
Drug Discovery: By revealing the precise 3D structures of proteins, cryo-EM can assist in drug design by identifying potential binding sites for therapeutic molecules. This has a direct impact on developing treatments for diseases like cancer and viral infections.
Viral Structures: Cryo-EM has been instrumental in visualizing virus particles, including structures of enveloped viruses like coronaviruses. This has furthered our understanding of virus-host interactions and informed vaccine development.

The Technology Behind Cryo-EM

Modern cryo-EM technology depends on several critical innovations that make it possible to achieve such high-resolution images and detailed structures:

JEOL CRYO ARM™ Microscopes: JEOL’s CRYO ARM™ series, including the CRYO ARM™ 200 and 300, offers highly stable cryo-electron microscopes designed for structural biology. These instruments combine extreme mechanical stability with automated data collection to produce consistent and high-resolution images.
Direct Electron Detectors: Direct electron detectors capture images with improved signal-to-noise ratios, enhancing the overall quality of the data. These detectors are essential for capturing the fine details of biological molecules.
Automated Data Collection: Software-driven automation allows researchers to image thousands of particles in a single experiment efficiently. This high-throughput capability significantly accelerates the pace of data collection, enabling researchers to solve structures more quickly.
Advanced Image Processing: Cryo-EM relies on sophisticated algorithms that align and average thousands of 2D images to reconstruct the 3D structure. Continuous advancements in image processing software have enhanced the resolution and accuracy of these reconstructions.

Explore JEOL’s Cryo-EM Solutions

Single-particle cryo-EM is revolutionizing how we understand the structure and function of biological molecules. With advances in cryo-electron microscopy technology, such as JEOL’s CRYO ARM™ series, researchers are now able to solve increasingly complex biological structures, paving the way for new discoveries in fields ranging from biochemistry to drug design.

JEOL’s state-of-the-art cryo-electron microscopes are designed to push the boundaries of what’s possible in structural biology. Visit our Cryo-EM Product page to learn more about our CRYO ARM™ series and how they can benefit your research.

Using Cryo-EM to Determine the Structure of Macromolecular Complexes

Cryo-electron microscopy (cryo-EM) transforms the way we visualize biological molecules. The ability to discern biological structures preserved in their natural state at near- or sometimes true atomic resolution gives scientists invaluable insights into the function of large and also dynamic macromolecules. Unlike X-ray crystallography, which requires crystallization, cryo-EM can be used on flexible molecular assemblies, allowing researchers to study complex biological systems that would otherwise remain out of reach. Success in cryo-EM largely depends on the preservation of biological molecules in a state as close to their native environment as possible. This advantage makes cryo-EM indispensable in revealing molecular interactions that are crucial for understanding biological processes.

Single Particle Analysis: Reconstructing 3D Structures

Single particle analysis (SPA) is a cryo-EM method used to determine the structure of vitrified biomolecules. This technique involves collecting multiple 2D projections of ideally randomly oriented particles. Advanced image processing software aligns these images and computationally reconstructs a detailed 3D structure. An advantage of SPA lies in its ability to capture various conformations of the same molecule, making it particularly useful for studying dynamic complexes. Moreover, sophisticated algorithms help handle challenges like sample heterogeneity and molecular flexibility, allowing scientists to obtain high-resolution structures of otherwise challenging biomolecules.

Advantages Over Traditional Methods

Cryo-EM offers several distinct advantages over other structure determination techniques such as X-ray crystallography and nuclear magnetic resonance (NMR). As mentioned above, cryo-EM does not require crystallization. Additionally, cryo-EM provides a more realistic depiction of biological molecules in their near-native environment. It can also capture macromolecules in multiple conformational states, offering insights into their functional dynamics. We'll discuss this more shortly. Also, cryo-EM can yield detailed structures from tiny amounts of material, often no more than a few micrograms.

Compared to NMR, cryo-EM can handle much larger complexes, as NMR struggles with molecules over 100 kDa due to signal overlap and complexity. These advantages have made cryo-EM an essential tool for structural biologists aiming to study complex biological systems, such as membrane proteins and other large assemblies.

How Does Cryo-EM Handle the Heterogeneity of Macromolecular Complexes?

As mentioned, cryo-EM's ability to handle the inherent heterogeneity of macromolecular complexes is a significant advantage. Biological molecules often exist in multiple conformational states, which can result in structural flexibility and variation. This heterogeneity presents challenges for researchers aiming to achieve high-resolution structures, but cryo-EM offers several computational and experimental strategies to manage this complexity.

Computational Approaches: Cryo-EM employs advanced computational techniques to manage heterogeneity. 3D classification algorithms sort particles into groups based on structural similarities, allowing for the reconstruction of multiple structures from within the same dataset. This technique is routinely used to isolate distinct conformational or compositional states. Masking techniques also allow researchers to focus within specific regions of a structure and improve the resolution of those regions while ignoring the signal from the more flexible parts. Finally, machine learning algorithms are increasingly being used to improve classification of heterogeneous data.
Experimental Approaches: Experimental methods such as biochemical optimization and ligand binding can also help reduce heterogeneity by stabilizing specific functional states. By refining sample preparation techniques, researchers can create more homogeneous conditions, reducing the impact of structural variability on the final reconstruction.

Together, these strategies enable cryo-EM to handle complex, flexible biological systems, offering a comprehensive view of macromolecular dynamics and the biological processes they regulate.

Challenges and Future Directions

Despite its many advantages, cryo-EM is not without its challenges. Sample preparation remains a critical step, with ongoing research focused on optimizing EM grid preparation and vitrification techniques to improve sample quality. Additionally, while cryo-EM has achieved remarkable resolution with large complexes, it faces limitations when applied to smaller proteins (less than 100 kDa). Addressing these challenges may involve further advances in detector sensitivity, image processing algorithms, and microscope hardware.

Nevertheless, the future of cryo-EM is promising as researchers continue to push the boundaries of what can be achieved with this technique. As technology improves, cryo-EM is expected to extend its reach into smaller proteins and more complex biological systems, further cementing its role as a cornerstone of structural biology and drug discovery.

Explore our site to learn more about the pioneering principles of cryo-EM, including how it differs from TEM. If you have any questions, contact a member of the JEOL team today.

References and further reading

Azinas S, Carroni M. Cryo-EM uniqueness in structure determination of macromolecular complexes: A selected structural anthology. Curr Opin Struct Biol. 2023 Aug;81:102621. doi: 10.1016/j.sbi.2023.102621. Epub 2023 Jun 12. PMID: 37315343.
Holger Stark, Ashwin Chari, Sample preparation of biological macromolecular assemblies for the determination of high-resolution structures by cryo-electron microscopy, Microscopy, Volume 65, Issue 1, February 2016, Pages 23–34, https://doi.org/10.1093/jmicro/dfv367
Carroni M, Saibil HR. Cryo electron microscopy to determine the structure of macromolecular complexes. Methods. 2016 Feb 15;95:78-85. doi: 10.1016/j.ymeth.2015.11.023. Epub 2015 Nov 27. PMID: 26638773; PMCID: PMC5405050.
Jonić S. Cryo-electron Microscopy Analysis of Structurally Heterogeneous Macromolecular Complexes. Comput Struct Biotechnol J. 2016 Oct 14;14:385-390. doi: 10.1016/j.csbj.2016.10.002. PMID: 27800126; PMCID: PMC5072154.

A Walkthrough of the Cryo-EM Workflow

Cryo-electron microscopy (cryo-EM) has truly revolutionized structural biology, giving scientists the power to explore the intricacies of macromolecular complexes at near-atomic levels. This technology is rapidly gaining ground over traditional X-ray crystallography, thanks to the advancements we're making at JEOL. In this blog, we'll walk you through the stages of the cryo-EM workflow, highlighting how our cutting-edge systems— the CRYO ARM™ —are paving the way for new discoveries in macromolecular atomic-level imaging.

Sample Preparation

Sample Production & Purification

Once a protein of interest is chosen, it typically needs to be expressed in cells and then purified. Protein purification begins with cell lysis, where cells are broken up to release their contents, followed by clarification to remove debris via centrifugation or filtration. The target protein is then captured using affinity chromatography, which selectively binds it. Further intermediate purification steps, such as ion exchange or hydrophobic interaction chromatography, enhance purity by separating proteins based on charge or hydrophobicity. Polishing is performed using size-exclusion chromatography to achieve final purity. The purified protein is then validated through SDS-PAGE, Western blot, or mass spectrometry before being stored under optimal conditions to maintain stability.

Grid Preparation & Vitrification

Grid preparation and vitrification are crucial steps in cryo-electron microscopy (cryo-EM). A small volume of protein sample is applied to a glow-discharged EM grid, typically coated with a thin carbon or gold support film with small holes that absorb the sample. Excess liquid is blotted away, leaving a thin layer of sample in the holes. The grid is then rapidly plunged into liquid ethane at cryogenic temperatures, a process called vitrification, which prevents ice crystal formation and preserves the sample in an amorphous, glass-like state. This ensures structural integrity for high-resolution imaging under an electron microscope. As this is crucial and heavily dependent on chemistry at fairly poorly understood interactions at the nano-scale, variations in freezing equipment and sample applications have been developed. For instance, using inkjet-like devices or piezo-electric nebulizers, precise quantities of sample can be applied. Application of the sample and the time that elapses before freezing is hugely critical; given the detrimental aspects of the air-water interface.

Microscopy

Grid Screening

In the initial stages of the cryo-EM workflow, screening can be performed using negative stain at room temperature or cryo-EM to assess sample quality. For negative staining, the protein sample is applied to a carbon-coated grid, stained with heavy metal salts (e.g. uranyl acetate), and air-dried, providing contrast for low-resolution imaging. This helps evaluate particle distribution, aggregation, and structural integrity. Alternatively, cryo-EM screening involves imaging vitrified samples to check for suitable particle concentration and ice thickness. This step ensures that only well-behaved samples proceed to high-resolution data collection on more advanced microscopes. Both negatively stained or vitrified grids can be screened with high contrast at 120 kV in the recently released JEOL JEM-120i. This lets the operator quickly assess the suitability of the sample for further, more demanding imaging. Our system’s design minimizes user strain, allowing quick evaluation and easy selection of the best samples for further studies.

Data Acquisition

Data collection for cryo-EM involves several key steps to obtain high-resolution images of vitrified biological samples. The process begins with grid loading into the JEOL CRYO ARM™ via its automatic sample loader, the cryoSPECPORTER™, which facilitates storage of up to 12 samples, and quick transfer to the microscope’s cryogenic sample stage. Before data acquisition, the sample may be screened under low-dose conditions to assess ice thickness and particle distribution, minimizing radiation damage. High-resolution images are then recorded using a direct electron detector (DED), capturing dose-fractionated movies to enhance the signal-to-noise ratio and help correct for beam-induced motion. Automated data collection software, such as SerialEM or JADAS, optimizes imaging conditions by accurate hole targeting and maintaining consistent defocus values. Additionally, image shift-based acquisition significantly increases throughput by reducing stage movements. This automation enables the collection of thousands of images while allowing the operator to perform parallel data analysis, thus streamlining the workflow.

Structure Determination

Image Processing

Cryo-EM data processing involves several steps to extract high-resolution structural information from raw images. The process begins with motion correction, where frames from dose-fractionated micrographs are aligned to correct for beam-induced motion. Next, CTF (Contrast Transfer Function) correction is applied to address imperfections in the electron microscope’s optics. Afterward, particle picking identifies and extracts individual particles from the micrographs. These particles are then classified into groups based on their orientations using 2D classification. An initial 3D model is generated from selected 2D classes and iteratively refined to improve resolution. The refinement process involves orientation assignment, angular refinement, local resolution assessment and even possible re-classification of particles. High-precision 3D refinement tools are compatible with validation tools, allowing for confident verification of the 3D models. By continually iterating on the refinement, high-resolution maps can be achieved that withstand rigorous scientific scrutiny.

Analysis and Interpretation

With map fitting and visualization tools, you’ll build accurate structures and gain enhanced understanding of molecular interactions, helping you unlock the biological significance of your findings. The tools make it easy to export and share your data with compatible formats for EMDB and PDB submissions, so you can confidently share your work with the scientific community.

At JEOL, we’re committed to helping you push the boundaries of what’s possible in structural biology. From sample preparation to final analysis, our experts and tools provide the support you need for groundbreaking research. To see how our JEM-120i and CRYO ARM™ can transform your workflow, reach out to us today.

With JEOL by your side, you can navigate the complexities of cryo-EM with ease and precision. Ready to elevate your research? Explore how JEOL’s technology can be part of your success in structural biology.

What is Transmission Electron Microscopy?

Transmission Electron Microscopy (TEM) is a powerful imaging and analytical technique, enabling scientists to visualize the internal structure of materials at atomic resolutions. Instruments like JEOL’s Transmission Electron Microscopes (TEM) allow researchers to analyze composition, morphology, and structure at nanoscale levels. This whitepaper explores the principles behind TEM, its core components, and its diverse applications across research and industry. Our TEMs are pivotal in fields such as materials science, life sciences, and nanotechnology due to their capability to reveal internal features with unmatched clarity, surpassing other imaging methods.

Principle and Operation of Transmission Electron Microscopy

Transmission Electron Microscopy relies on the transmission of a high-energy electron beam through an ultra-thin sample (typically less than 100 nm thick) to produce high-resolution images. Electrons, due to their extremely short wavelengths, can resolve fine structural details that are inaccessible to light-based microscopy. Unlike Scanning Electron Microscopy (SEM), which provides surface imaging, TEM generates an in-depth view of the internal structure of the specimen. The transmitted and scattered electrons produce a detailed image that reveals both structural and compositional information at atomic levels.

How TEM Works

1. Electron Source and Beam Generation

TEM instruments use an electron gun to emit a coherent beam of electrons, typically via thermionic or field emission. Thermionic emission occurs when a heated tungsten filament or lanthanum hexaboride (LaB₆) rod releases electrons, whereas field emission uses a sharp tungsten tip to generate electrons under high electric fields. Our JEM-2100Plus utilizes a LaB6 source, while our cold-field emission gun (CFEG), found in models like the JEM-F200 and ARM series, generates high brightness and energy resolution, suitable for high-resolution imaging where low chromatic aberration is beneficial. Each electron source is optimized to match specific application needs, whether for life sciences or materials research.

This electron beam is accelerated to high energies (usually 80 to 300 keV), which allows the electrons to penetrate a thin sample.

2. Interaction with the Sample

The specimen used in TEM must be extremely thin—ideally less than 100 nm—to allow electrons to pass through without significant absorption. The interaction between the electron beam and the sample results in scattering, diffraction, and transmission of the electrons, producing contrast and structural information in the resulting image. Electrons that pass through the sample without scattering form the bright field image, whereas electrons scattered at specific angles can be used for dark field imaging.

Comparative Analysis with Other Microscopy Techniques

TEM provides significantly higher resolution than traditional light microscopy because the wavelength of accelerated electrons is many times shorter than visible light, governed by the de Broglie wavelength equation. JEOL’s GRAND ARM™2, for example, achieves resolutions under 60 pm, capturing atomic-scale details. Magnifications can reach up to 50 million times, making TEM one of the most powerful tools for studying atomic structures.

TEM offers unique advantages in imaging the internal structure of samples at atomic resolution, but other microscopy techniques have distinct features that make them suitable for different applications:

Scanning Electron Microscopy (SEM): Unlike TEM, which provides internal structural information, SEM scans the surface of a sample to produce 3D-like images, ideal for surface morphology studies.
Atomic Force Microscopy (AFM): AFM offers nanometer resolution without requiring a vacuum or conductive coating. It can provide information on surface topology that complements TEM data.
X-ray Microscopy: X-ray microscopy enables imaging of thicker samples in their native state, which TEM cannot easily do due to sample thickness limitations. This comparison highlights when TEM is the preferred tool for atomic-level detail.

Components of a JEOL TEM

JEOL TEMs integrate a range of subsystems, from electron sources to lens systems, to provide versatile imaging solutions. Key components include:

Specimen Stage: The stage is optimized for precise positioning, with some models offering cryogenic capabilities, as in JEOL’s CRYO ARM™ Series. This is essential for imaging electron beam-sensitive biological specimens in their native state.
Imaging System: JEOL TEMs, like the JEM-F200, combine objective, intermediate, and projector lenses to magnify the sample image to atomic resolutions. These images can be viewed on a fluorescent screen, a CCD camera, or other detectors.
Vacuum System: Maintaining a high vacuum (typically below 10⁻⁵ Pa) prevents electron scattering, critical for clear image quality. JEOL systems use a combination of roughing pumps, turbomolecular pumps, and ion getter pumps to maintain stable vacuum levels essential for high-resolution imaging.

Challenges in Sample Preparation and Solutions

Sample preparation is often a complex and delicate process for TEM, particularly due to the requirement for ultrathin specimens. Specific challenges include:

Ion Beam Damage during FIB Milling: Focused Ion Beam (FIB) milling can introduce damage to the sample surface, affecting the integrity of the observed features. Using low-dose FIB techniques and cryogenic FIB (cryo-FIB) can help mitigate damage.
Artifacts in Biological Samples: Staining biological samples with heavy metals like osmium tetroxide may introduce artifacts that obscure genuine structures. Cryo-preservation offers an alternative to staining, preserving samples in their native state with minimal chemical alteration.

Addressing these challenges through innovative sample preparation methods can make TEM more approachable and minimize potential sample damage or artifacts.

Interpreting TEM images accurately requires not only skill but also advanced post-processing techniques. Fourier transform filtering can be used to enhance image quality, helping to distinguish between real features and noise. Techniques such as contrast adjustment are also essential for bringing out subtle differences in electron density. Common artifacts, such as beam damage or astigmatism, can lead to misinterpretation if not properly accounted for. JEOL provides specialized training and software tools to assist users in effective image interpretation, ensuring reliable data.

Sample Preparation

The preparation of TEM samples is one of the most challenging aspects of the technique due to the need for ultrathin specimens. Methods include:

Ultramicrotomy: Used for biological samples and soft materials, where a diamond or glass knife cuts the sample into ultrathin slices (50-100 nm).
Focused Ion Beam (FIB) Milling: Common in materials science, FIB uses a gallium ion beam to thin specific regions of a sample. This method is precise but can introduce some ion beam damage.
Cryo-Preparation: Cryo-TEM is used for biological samples to preserve their native structure. The sample is vitrified by rapid freezing to avoid ice crystal formation, providing a snapshot of the sample in its natural state.
Staining with Heavy Metals: Biological samples are often stained with heavy metals such as osmium tetroxide or uranium acetate to enhance contrast, as organic materials have low electron density.

Types of TEM Imaging

Bright Field Imaging: The most common imaging mode, using unscattered electrons to create contrast between different regions of the sample.
Dark Field Imaging: Utilizes scattered electrons to form the image, which enhances contrast for specific features like crystalline defects or inclusions.
Electron Energy Loss Spectroscopy (EELS): An advanced technique where energy losses experienced by electrons interacting with the sample are measured, providing detailed information on chemical composition, bonding, and electronic structure. EELS is particularly effective for analyzing light elements and understanding complex bonding states. For example, EELS can be used to study the oxidation states of transition metals in catalysts or to determine the presence of specific functional groups in polymers.
Energy-Dispersive X-ray Spectroscopy (EDS): EDS allows for elemental analysis by detecting characteristic X-rays emitted from the sample. It is particularly useful in mapping the distribution of elements within a sample, which helps in the study of material composition, inclusions, and impurities. In combination with EELS, EDS provides a comprehensive analysis of both light and heavy elements within the sample.

Applications of JEOL TEM Systems

Materials Science: TEM is instrumental in studying microstructures, phase boundaries, dislocations, and other crystal defects. It provides insights into mechanical properties, thermal stability, and phase transformations, making it essential for materials development and failure analysis.
Life Sciences: In biological research, TEM is used to visualize organelles, viruses, and protein complexes at high resolution. JEOL’s CRYO ARM™ Series has revolutionized structural biology by allowing researchers to determine the 3D structures of biomolecules, providing capabilities for single-particle analysis and tomography with high throughput and precision.
Nanotechnology: Characterization of nanomaterials, such as nanoparticles, nanotubes, and nanowires, is a major application. TEM provides detailed information on size, shape, and structure, which is critical for understanding their physical and chemical properties.
Semiconductor Research: TEM is employed in the semiconductor industry to examine layer thicknesses, grain structures, and defects in microelectronic devices. It plays a crucial role in quality control and the development of new electronic materials.
Paleontology and Palynology: TEM helps in the study of fossilized organic material and spores, providing valuable insights into past environmental conditions and evolutionary biology.

Advantages and Limitations

Advantages: TEM offers extremely high resolution, allowing imaging at atomic scales. It provides both structural and compositional information through techniques such as Energy-Dispersive X-ray Spectroscopy (EDS) and Electron Energy Loss Spectroscopy (EELS). Its versatility makes it applicable across numerous scientific disciplines.
Limitations: TEM has several limitations, including the need for very thin samples, which often requires complex and time-consuming preparation. Additionally, the vacuum environment can be challenging for certain biological samples, and the high-energy electron beam can cause damage to sensitive materials. The equipment itself is expensive, and the operation requires highly skilled personnel.

Interested in TEM Imaging?

JEOL's Transmission Electron Microscopy systems are vital tools in modern science, offering unmatched capabilities for the visualization and analysis of materials at atomic levels. Despite its challenges, advances in sample preparation, imaging technologies, and analytical techniques continue to push the boundaries of what TEM can achieve. It remains at the forefront of research and development across materials science, nanotechnology, and biology, providing essential insights into the fundamental nature of materials and biological systems.

Field Operation Solutions: JEOL’s factory-certified engineers, averaging 20+ years of experience, ensure precision installation, maintenance, and repair services.
Dedicated Technical Support: A Service Support Group provides model-specific assistance, ensuring quick resolutions to any technical issues.
Service Level Agreements: Tailored service packages cover maintenance and repairs to maximize instrument longevity.
Comprehensive Training: Through hands-on courses, JEOL helps users fully leverage the potential of their TEMs.

For more in-depth information on Transmission Electron Microscopy, refer to our additional resources on materials science and structural biology. If you're keen to learn more about specific instrument suitability for your unique use case, contact a member of the JEOL USA team today.

Cryo-EM vs. X-ray Crystallography

Cryo-electron microscopy (cryo-EM) and X-ray crystallography are two essential techniques for enhancing our understanding of biological macromolecules at atomic resolution. Though distinct in methodology and application, these techniques are highly complementary, with each filling gaps left by the other. As researchers go deeper into the complex mechanisms underlying biological processes, leveraging both methods offers unparalleled insights, pushing structural biology toward new heights of discovery.

X-ray Crystallography: The Gold Standard

Historically, X-ray crystallography has been the predominant technique in structural biology. In this method, an X-ray beam passes through a crystallized sample and a diffraction pattern is created when the X-rays interact with the well-ordered crystal lattice. By analyzing the Bragg reflections in these patterns in terms of amplitudes and phases researchers can reconstruct the molecule's three-dimensional structure typically with atomic precision. The sharpness of these diffraction spots is key, directly correlating to the quality and order of the crystal. However, completeness of the reflections in each resolution zone plays another essential role in determining the final resolution of the three-dimensional structure and thus to what extent the structure can be interpreted.

However, a notable challenge is that not all biomolecules are easy to crystallize. Many biologically significant structures, such as membrane proteins or large, flexible macromolecular complexes, resist crystallization due to their dynamic nature. Also, crystallization often requires significant sample quantities and molecular engineering to stabilize flexible regions—limitations that have sparked the search for alternative methods. Furthermore, intermediary structures that can provide snapshots of important dynamic processes are extremely hard to crystallize.

Despite these hurdles, X-ray crystallography remains unsurpassed in resolving fine atomic details of well-ordered macromolecules. Its precision makes it the go-to technique for studying stable and crystallizable proteins, enzymes, and other biomolecules. Structural biologists use it to uncover mechanistic details that are crucial for drug development and understanding molecular interactions.

Cryo-Electron Microscopy: Flexibility for Non-Crystalline Samples

Cryo-EM, by contrast, doesn't require crystallization. Instead, biological samples are flash-frozen, capturing molecules in their near-native state. A high-energy electron beam is passed through these frozen samples, producing 2D projections of said molecules at various orientations. These images are then computationally assembled into a 3D map of the molecule.

Cryo-EM has surged in popularity, especially for large macromolecular complexes that are difficult or impossible to crystallize. Recent advancements in cryo-EM technology—such as improved electron detectors and image-processing algorithms—have led to what is referred to as the "resolution revolution." With cryo-EM, researchers can now achieve near-atomic resolution for even flexible or heterogeneous assemblies, such as viruses, ribosomes, and protein complexes.

One of cryo-EM's primary strengths is its ability to visualize large, multi-component structures in different conformational states. This flexibility enables scientists to capture dynamic interactions within macromolecular machines, something X-ray crystallography struggles to achieve. However, cryo-EM's resolution typically doesn't match the atomic-level precision of crystallography, particularly for smaller proteins or structures below 100 kDa.

Complementary Roles in Structural Biology

Despite their differences, cryo-EM and X-ray crystallography are not competing technologies but rather complementary tools that can work together for more complete structural insights. One of the most practical synergies between these methods is solving structures through an integrated approach.

For instance, cryo-EM can generate an initial low- to medium-resolution 3D map of a large protein complex, providing the overall architecture. X-ray crystallographic data of individual components can then be docked into the cryo-EM map, revealing high-resolution details of specific domains or subunits within the larger complex. This combined approach has been particularly valuable in studying systems that are too dynamic or heterogeneous for crystallography alone. Well before the so-called ‘resolution revolution’ this was effectively the go-to technique in the 1980s to elucidate large complexes at resolution beyond a nanometer.

Conversely, cryo-EM can assist X-ray crystallography by solving one of its most notorious hurdles—the phase problem. X-ray diffraction data capture only the intensity of the Bragg wave, but not its phase, making it impossible to resolve structures of large biomolecular complexes without a suitable means to derive these phases. Crystals of small compounds, such as paraffins, can often be solved using direct methods. These types of samples are also extremely well-suited to be solved using microED. Cryo-EM maps can provide an initial model for molecular replacement, thus enabling researchers to determine phases and solve the crystal structure at higher resolution.

Additionally, the distinct physical principles underlying the two methods—X-ray photons interacting with electron clouds in crystallography versus high-energy electrons interacting with atomic Coulomb potentials in cryo-EM—mean they provide slightly different structural information. In practice, this allows researchers to study molecules in different states or conformations, offering a more holistic view of biological structure and function.

Future Directions: Integration for Enhanced Discovery

As both cryo-EM and X-ray crystallography technologies continue to advance, the lines between them may blur further, with researchers increasingly using both techniques in tandem to solve complex structural puzzles. With cryo-EM achieving near-atomic resolution and micro-electron diffraction (microED) emerging as a method to solve crystallography-style biomolecules with very small crystals, structural biology is poised for breakthroughs that were unimaginable just a few decades ago.

JEOL has been at the forefront of this evolution, providing state-of-the-art cryo-EM systems that offer unmatched resolution and flexibility. By integrating cryo-EM with X-ray crystallography data, researchers using JEOL instruments can push the boundaries of structural biology, revealing intricate biological systems in unprecedented detail.

Ultimately, leveraging both cryo-EM and X-ray crystallography allows scientists to investigate macromolecular structures with an accuracy and completeness that no single method can achieve alone. As structural biology progresses, the combination of these complementary techniques will continue to drive innovation and discovery across the life sciences.

References & Further Reading

Wang HW, Wang JW. How cryo-electron microscopy and X-ray crystallography complement each other. Protein Sci. 2017 Jan;26(1):32-39. doi: 10.1002/pro.3022. Epub 2016 Sep 7. PMID: 27543495; PMCID: PMC5192981.

Which Techniques are Used in Lithium-Ion Battery Analysis?

In modern technology, lithium-ion batteries (LIB) are found in many different applications, including electric cars and portable electronic devices, such as tablets and smartphones. Other purposes include aerospace and defense industries, energy storage systems, and medical devices. Lithium-ion batteries are now considered essential for many technological applications, making their analysis vital for manufacturing, enhancement, and applications. This blog post will focus on the techniques used to analyze lithium-ion batteries.

Why Do We Analyze Lithium Ion Batteries?

Lithium-ion batteries consist of many different components, layers, and structures that are essential for their high-performance properties. These comprise of fluids, powders, sheets, and other materials. The importance of analyzing LIBs lies in understanding their quality and reliability, which impacts their uses in various industries. Analyzing lithium-ion batteries is necessary to understand how they age, what internal changes occur, and what properties are present.

Lithium Ion Batteries Analysis Techniques

There is a growing demand for high-performance, durable lithium-ion batteries, especially now that their applications span multiple industries. Due to technological and scientific advances, several methods can be used to analyze lithium-ion batteries and their components. Below, we provide a brief overview of some key methods:

Scanning Electron Microscopy (SEM): SEM is a widely used method for studying the fine surface and internal structures and chemical properties of battery materials. It is also helpful for monitoring reactions and performance in next-generation batteries. Moreover, when coupled with a Windowless Energy Dispersive X-ray detector (EDS), this instrument is capable of observing lithium.
Transmission Electron Microscopy (TEM): This method helps scientists monitor the microstructural characteristics of lithium-ion batteries, including electrodes and materials, and study chemistry between battery components.
Auger Microprobe (EMAS): Used to detect lithium, analyze a sample's surface and internal regions, and conduct depth profiling of positive and negative electrode materials, key components in lithium batteries. EMAS is also used to analyze chemical processes, irregularities, and surface structures of LIBs.
X-ray Fluorescence Spectroscopy (XRF): XRF enables the analysis of types and concentrations of elements in a sample, which includes the powder used in lithium-ion batteries. This method is used to identify contaminants and the structural composition of LIBs.

JEOL USA: Lithium Ion Batteries

JEOL USA offers the instruments discussed above and more for the analysis of lithium-ion batteries. By using these tools, engineers and researchers can assess the performance and safety levels of these systems, leading to the manufacture of higher-quality products.

JEOL solutions are ideal for analyzing the performance and quality of LIBs, from manufacturing and failure analysis to research and development. The product ranges include spectrometers, microanalyzers, electron microscopes, and other scientific systems.