Site Selection & Protection: Use SEM imaging to locate the area of interest on the sample. Deposit a protective layer (usually platinum or carbon) over this site to shield it during ion milling.
Coarse Milling: Mill away material around the protected site, creating a thin slab (lamella) that remains attached at one end for support. This isolates a small section containing the region of interest.
Lift-Out & Mounting: Attach a nanomanipulator probe to the lamella (often using ion-beam-deposited platinum) and cut it free from the bulk. Lift the lamella out and weld it onto a TEM grid or holder, then detach the probe.
Final Thinning & Polishing: Thin the lamella from both sides to make it electron-transparent (typically <100 nm thick). Perform a final low-energy polish to remove any damaged layer and reach the desired final thickness (~30 nm). The sample is now ready for TEM imaging.

Modern FIB-SEM systems can automate much of this workflow. Many offer predefined milling recipes and in-situ monitoring (e.g., a STEM detector inside the FIB-SEM) to check lamella quality, making the process more efficient and user-friendly.

Applications Across Industries

FIB-SEM-based TEM sample preparation is used in many fields due to its versatility and precision. Key areas include:

Semiconductors: The semiconductor industry relies on FIB-SEM to extract site-specific cross-sections (like a single transistor or interconnect) for TEM imaging, allowing engineers to pinpoint defects or verify structures in advanced chips that are impossible to isolate otherwise.
Materials Science: Materials researchers use FIB-SEM to prepare TEM samples from specific microstructural features – such as a grain boundary, a precipitate, or a phase interface. This precision is valuable for studying localized phenomena (corrosion layers, nanoscale precipitates, deformation zones) in metals and ceramics.
Life Sciences: Biologists use FIB-SEM, often under cryogenic conditions, to thin specific regions of cells or tissues for TEM, enabling 3D visualization of internal structures (organelles, viruses) at nanometer resolution via cryo-electron tomography. This method complements traditional ultramicrotomy by allowing targeted thinning without mechanical slicing.

Why Choose JEOL?

(JEOL UK News | Launch of the FIB-SEM System "JIB-PS500i" with Hig) The JEOL JIB-PS500i FIB-SEM system provides an all-in-one solution for fast, precise TEM sample preparation, integrating high-current ion milling and high-resolution SEM imaging in a single instrument.

JEOL has long been at the forefront of electron microscopy, and this expertise extends to FIB-SEM technology. A prime example is the JEOL JIB-PS500i – a state-of-the-art FIB-SEM system for fast, precise TEM sample preparation. This instrument delivers the synergy of rapid ion milling, high-resolution SEM imaging, and in-situ EDS analysis in one platform.

The JIB-PS500i emphasizes high-quality, rapid prep. It can produce ultra-thin lamellae (often <30 nm) suitable for atomic-resolution TEM, and it includes a retractable STEM detector to check specimen quality during milling. With features like the TEM-Linkage double-tilt holder, users can transfer a prepared lamella directly from the FIB-SEM to a JEOL TEM without air exposure, minimizing contamination. The system also offers automated preparation routines (JEOL’s STEMPLING™) that make consistent results achievable even for newer operators.

JEOL’s FIB-SEM is built for flexibility and throughput. A large chamber and 5-axis stage accommodate diverse sample sizes, and a high-current ion beam (up to 100 nA) enables fast bulk milling – especially useful for semiconductor workflows. At the same time, the instrument excels at low-current fine polishing for delicate samples. This all-in-one capability means one machine can handle everything from coarse cross-sectioning to final thinning. With JEOL’s strong support network backing the technology, users can confidently elevate their TEM sample preparation using these advanced tools.

Closing Thoughts

TEM sample preparation is a critical step that directly impacts the quality of results. Overcoming challenges of thickness, cleanliness, and damage is key to unlocking the full power of TEM’s atomic-level imaging. Focused Ion Beam-SEM technology has emerged as a definitive solution, enabling consistent production of ultra-thin, high-quality samples across diverse applications. What was once a daunting, time-consuming task can now become a streamlined routine in the lab.

JEOL’s commitment to advanced sample prep exemplifies how modern tools make this process more reliable and efficient. By pairing sound preparation practices with FIB-SEM instrumentation, users can obtain clearer images and more accurate data from their TEM analyses. In short, better sample prep leads to better science. We encourage you to explore JEOL’s FIB-SEM solutions to elevate your TEM work.

Structural biology 101: Principles, techniques and applications

The molecular machinery of life operates with astonishing precision. Proteins fold into intricate geometries, nucleic acids assemble into double helices and tertiary structures, and multi-subunit complexes carry out processes fundamental to cellular survival. Understanding these structures, and how they determine biological function, is the domain of structural biology.

It is not enough to know what molecules are made of. We must comprehend how they are built.

The Central Premise: Structure Determines Function

Structural biology rests on a fundamental axiom of molecular life sciences: the three-dimensional shape of a biomolecule dictates its biological role. From enzymatic catalysis and signal transduction to genome maintenance and immune recognition, structural conformation governs interaction, specificity, and activity.

Even minor perturbations in structure, such as point mutations, deletions, or misfolding, can compromise function, drive pathogenicity, or confer drug resistance. In this context, elucidating structure is not merely descriptive; it is diagnostic, predictive, and, increasingly, design-oriented — making structural biology a critical tool in modern molecular research.

Molecular Architecture: Hierarchical and Informative

Biological macromolecules exhibit a multi-tiered architecture:

Primary structure: Linear sequences of amino acids (proteins) or nucleotides (DNA/RNA).
Secondary structure: Localized folding motifs, including α-helices, β-sheets, and loops, stabilized by hydrogen bonds.
Tertiary structure: The complete three-dimensional conformation of a single polypeptide or nucleic acid strand, shaped by hydrophobic interactions, ionic bridges, disulfide bonds, and van der Waals forces.
Quaternary structure: The spatial organization of multiple subunits into higher-order complexes, often essential for cooperative function.

This structural framework enables researchers to interpret molecular behavior, predict function based on form, and design biological systems with enhanced or novel capabilities.

Techniques in Focus: Methods for Determining Molecular Structure

Structural biology relies on a suite of experimental and computational techniques to determine the architecture of macromolecules with precision and clarity. Each method offers distinct advantages, and together, they provide a comprehensive view of biological structure and function.

X-ray Crystallography

A cornerstone of structural biology, X-ray crystallography involves directing X-rays through a crystallized biomolecule and analyzing the resulting diffraction pattern. Using a variety of techniques to modify the original diffraction amplitudes so as to extract phases for each of the Bragg reflections enables the generation of high-resolution electron density maps and the construction of detailed atomic models. It is widely used to study enzymes, nucleic acids, and protein-ligand interactions, particularly where static, well-defined structures are needed for applications such as drug design and mechanistic elucidation.

Cryo-Electron Microscopy (Cryo-EM)

Cryo-EM allows scientists to visualize macromolecular complexes in near-native states by imaging specimens frozen in vitreous ice. Thousands of two-dimensional images are computationally aligned to reconstruct a three-dimensional structure. Its strength lies in resolving large and flexible biological assemblies, making it essential for studying membrane proteins, viral particles, and ribonucleoprotein complexes. Also, typically much smaller quantities of the biomolecule of interest can be used for a structural study using cryo-EM and image processing, colloquially known as SPA. Although not always reaching down to atomic resolution, SPA nonetheless has proven itself to be the promising technique over x-ray diffraction and NMR for resolving many important biomolecules. Thus, this method has become pivotal in comprehending structural heterogeneity and conformational dynamics in structural biology.

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR provides atomic-level insights into biomolecules in solution by measuring the magnetic properties of specific atomic nuclei. It is particularly effective for probing protein dynamics, conformational changes, and transient interactions that occur in a physiological environment. In structural biology, NMR is indispensable for characterizing smaller proteins and intrinsically disordered regions. The technique provides a complementary view to the models provided by crystallography or Cryo-EM.

MicroED

This technique is an equivalent of xray diffraction in that electrons are used to diffract off a crystalline sample. Since electrons interact so much more with matter than xray photons, much smaller crystals can be used in this technique. Applied to small molecules this technique has proven to be incredibly successful in solving structures with great precision and speed often in a matter of minutes.

Computational Modeling and AI-Based Prediction

Computational methods have become an essential component of structural biology, offering powerful tools for predicting molecular structures directly from sequence information. These approaches leverage biophysical principles and evolutionary relationships to generate models of three-dimensional conformation with growing precision. When integrated with experimental techniques, computational modeling helps interpret structural data, identify functionally relevant regions, and explore the effects of sequence variation. This synergy extends the reach of structure-based research, enabling a deeper mechanistic understanding and more informed molecular design.

Applications: Translational Insights and Technological Innovation

Structural biology informs a broad spectrum of scientific and industrial domains:

Structure-Based Drug Design (SBDD): Precise knowledge of target geometry enables rational ligand design, optimizing binding affinity and selectivity while reducing off-target effects.
Mechanistic Studies of Disease: Structural analysis elucidates how mutations destabilize protein folding, promote aggregation (e.g., in neurodegenerative disorders), or alter enzymatic activity.
Immunology and Vaccine Development: Structural insights into antigen presentation and epitope accessibility have accelerated monoclonal antibody engineering and the rational design of immunogens.
Synthetic and Systems Biology: De novo protein design, pathway reengineering, and the development of synthetic molecular machines all rely on structural characterization.
Environmental and Agricultural Biotechnology: Enzyme engineering for pollutant degradation or enhanced crop resilience often begins with structural templates.

Pioneering Structural Biology with JEOL USA

Structural biology offers a powerful framework for exploring the architecture of life at the molecular scale. Techniques such as Cryo-EM, X-ray crystallography, and NMR spectroscopy reveal the structures that govern biological activity, offering insights essential to fields ranging from drug discovery to synthetic biology.

As a global leader in scientific instrumentation, we, JEOL, support this work by providing high-performance tools tailored to the complex demands of structural biology. Our CRYO ARM™ series and advanced NMR spectrometers are designed to deliver the resolution, stability, and analytical precision required to decode molecular structures with confidence.

By equipping researchers with the means to visualize and interpret molecular form, we enable discovery at the atomic level— advancing not only scientific understanding but also broadening the impact of structural biology across research and innovation.

The role of structural biology in drug discovery

Successful drug discovery begins with comprehending the shape of biological molecules and how it affects their behaviour. Structural biology makes this possible by providing a detailed view of how targets function and where intervention is most likely to succeed. Its insights support every stage of development, helping researchers choose viable targets, design more effective compounds, and respond to challenges along the way. The connection between structure and strategy plays a central part in shaping modern therapies.

Structural Biology in Drug Discovery: Setting the Groundwork

Structural biology examines the three-dimensional form and flexibility of biological macromolecules such as proteins and nucleic acids. These molecular shapes are not fixed; they change in response to their environment and/or their specific activity and define how cells function or fail. In drug discovery and development, this information is far from theoretical. It helps determine if a molecule is accessible, stable enough for binding, and suitable for therapeutic intervention.

Strategic decisions often rest on this knowledge. By identifying molecular features that indicate accessibility or instability, structural data helps research teams refine their focus. Resources are channelled toward the targets with the highest potential for therapeutic success, improving both efficiency and direction.

How Structural Biology Shapes Drug Discovery

Decisions in drug discovery often depend on how well a target can be understood at the structural level. Whether evaluating a potential binding site or refining a compound’s fit, molecular detail shapes each step forward. The following sections show how structural detail supports key decisions throughout the drug discovery process.

Revealing Viable Targets

The first step in drug discovery involves pinpointing disease-related molecules that can be effectively targeted with therapeutic compounds. This evaluation benefits from a detailed structural view. It reveals characteristics such as pocket depth, surface contour, and stability that affect how well a drug can bind.

When mutations occur, they can change how a protein folds, how accessible its binding site is, or how stable the structure remains under physiological conditions. Identifying structural changes helps determine target suitability for drug development. Structural biology provides the clarity to do so.

Enabling Structure-Based Molecule Design

Once a target has been selected, the next stage is to craft a molecule that fits it with precision. Structural models help researchers identify key contact points and modify chemical groups to enhance binding, specificity, and stability. These refinements improve the quality of the drug candidate and reduce the number of iterations needed during development. Through this approach, drug discovery becomes more efficient. Researchers are able to narrow their focus to compounds that meet defined structural criteria, increasing the likelihood of successful outcomes in testing. Resolution at this point is key for the success of this step. As hydrogen-based interactions are often critical for the stability of drug binding in a protein pocket, generally speaking the higher the resolution the better.

Building from Fragment-Level Interactions

Drug discovery can begin with small molecular fragments that form initial connections with specific regions on a target. Although the binding is often subtle, careful structural mapping reveals valuable starting points for design. Early interactions help define a path toward building more complete molecules with stronger binding and better functional outcomes. Fragments that show potential can be chemically expanded or joined, a strategy that has opened new pathways to address targets previously considered too complex for conventional approaches.

Clarifying Mechanism and Functional Impact

A drug's ability to bind a target is only meaningful if it leads to the desired biological effect. Structural studies go beyond confirming attachment. They reveal whether the drug inhibits function, alters shape to block downstream signalling, or stabilizes a particular conformational state. Understanding how a compound affects target function ensures it performs as intended and provides the level of evidence needed to support clinical progression.

Anticipating and Addressing Resistance

Mutations that alter protein structure can quickly erode a drug’s effectiveness. Structural comparisons expose the points of divergence that impact binding, making it possible to redesign compounds that maintain performance. This adaptability is especially important in oncology and infectious disease, where resistance often limits long-term treatment value.

Structural Biology in Action: JEOL Tools for Drug Discovery

JEOL USA provides instrumentation that supports structure-based drug development from early investigation through to advanced refinement. Each tool is designed to resolve structural detail with clarity, helping researchers understand their targets and optimize how therapies interact with them.

Examples of JEOL USA’s structural biology instruments include:

The CRYO ARM Series: Enables high-resolution cryo-electron microscopy for large protein complexes and membrane proteins. Ideal for studying native-state structures without crystallization.
The JEM-F200 TEM System: Supports high-contrast imaging for biological screening and cellular structure analysis.
The 800 MHz NMR Spectrometer: Delivers detailed insights into small molecule and protein dynamics in solution. It's especially useful for fragment-based screening and conformational analysis.

Together, these instruments help researchers apply structural knowledge more effectively across each phase of drug discovery and development.

Advancing Drug Discovery Through Structural Clarity

Structural biology informs each part of the drug discovery process, where decisions often depend on a detailed understanding of molecular behaviour. Insight into target structure helps researchers align their strategies with biological reality. A clearer view of molecular structure ensures each state of development is grounded in evidence and aimed towards therapeutic success.

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.

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Is personalized structural biology a new frontier in medicine?

Is personalized structural biology a new frontier in medicine?

Turning Molecular Insight into Medical Precision

Why Personalized Structural Biology Represents a New Frontier in Medicine

From Genetic Code to Functional Clarity

Tailoring Therapies to Molecular Architecture

Anticipating and Overcoming Drug Resistance

Building the Foundation for Virtual Patients

Key Techniques Powering Tailored Insights

Enhancing Precision Medicine Through Personalized Structural Biology

Cryo-EM: Integrating Structural Bioinformatics for Functional Insights

Cryo-EM: Integrating Structural Bioinformatics for Functional Insights

Summary

Why Structural Bioinformatics Integration Enhances Cryo-EM

Preparing Cryo-EM Data for Structural Interpretation

Interpreting Cryo-EM Maps with Computational Tools

Supporting Complex Structural Analysis

Linking Structure to Function with Structural Bioinformatics

Data Quality and Instrumentation

Powering Integrated Structural Workflows with JEOL USA

An Overview of Different GC-MS Ionization Techniques

An Overview of Different GC-MS Ionization Techniques

The CI Process: A Closer Look

Advantages of CI

Alternatives to CI Techniques

Field Ionization (FI)

Photoionization (PI)

Electron Ionization (EI)

Closing Thoughts

Adhesives: Key Applications of Polymer Identification

Adhesives: Key Applications of Polymer Identification

Deformulation and Quality Control: A Precursor to Innovation

Enhancing Performance through Pyrolysis-GC-MS

Competitive Analysis and Product Development

Quality Control and Regulatory Compliance

Interested in Polymer Identification Tests?

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A Beginner's Guide to TEM Sample Preparation

A Beginner's Guide to TEM Sample Preparation

Challenges in TEM Sample Preparation

FIB-SEM: An Advanced Solution

Step-by-Step Workflow

Applications Across Industries

Why Choose JEOL?

Closing Thoughts

Structural biology 101: Principles, techniques and applications

Structural biology 101: Principles, techniques and applications

The Central Premise: Structure Determines Function

Molecular Architecture: Hierarchical and Informative

Techniques in Focus: Methods for Determining Molecular Structure

X-ray Crystallography

Cryo-Electron Microscopy (Cryo-EM)

Nuclear Magnetic Resonance (NMR) Spectroscopy

MicroED

Computational Modeling and AI-Based Prediction

Applications: Translational Insights and Technological Innovation

Pioneering Structural Biology with JEOL USA

The role of structural biology in drug discovery

The role of structural biology in drug discovery

Structural Biology in Drug Discovery: Setting the Groundwork

How Structural Biology Shapes Drug Discovery

Revealing Viable Targets

Enabling Structure-Based Molecule Design

Building from Fragment-Level Interactions

Clarifying Mechanism and Functional Impact

Anticipating and Addressing Resistance

Structural Biology in Action: JEOL Tools for Drug Discovery

Advancing Drug Discovery Through Structural Clarity

Understanding Radiation Damage in Cryo-Electron Microscopy

Understanding Radiation Damage in Cryo-Electron Microscopy