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A Beginner's Guide to TEM Sample Preparation

A Beginner's Guide to TEM Sample Preparation

Transmission Electron Microscopy (TEM) is a cornerstone technique in materials science, nanotechnology, and related fields. TEM reveals a material’s internal structure at near-atomic resolution by transmitting a high-energy electron beam through an ultra-thin specimen. This makes TEM indispensable for researchers and industry professionals who need to analyze materials in extreme detail. However, achieving high-quality TEM results starts long before the sample is loaded into the microscope. Sample preparation is critically important: only a properly thinned, clean, and undamaged specimen will yield clear and meaningful TEM images.

In this guide, we outline the fundamentals of TEM sample preparation and explore how modern Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has advanced this process to address common challenges.

Challenges in TEM Sample Preparation

Preparing a specimen for TEM is often as challenging as the imaging itself. The primary goal is to make the sample extremely thin (tens of nanometers or less) so that electrons can pass through it. If the sample is too thick, the electron beam is scattered or blocked, leading to poor image quality and lost detail. At the same time, the preparation process must avoid introducing damage or artifacts. Traditional polishing can bend or scratch the sample, and even FIB thinning can create an amorphous surface layer if done improperly. Such damage might produce micro-cracks or blurred structures that obscure true material features.

Another major concern is contamination. During preparation and transfer, samples can accumulate hydrocarbons or moisture from the air. In the TEM’s high vacuum, these contaminants are deposited under the electron beam, forming unwanted layers on the sample that mask real features. Careful handling and cleaning (e.g., plasma cleaning) are essential to minimize contamination. These challenges of thickness, damage, and contamination mean advanced preparation techniques are needed – and FIB-SEM has emerged as a leading solution.

FIB-SEM: An Advanced Solution

Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) combines two powerful capabilities in one instrument: precise ion milling and high-resolution SEM imaging. In a FIB-SEM, a gallium ion beam sputters away material while a coincident electron beam provides real-time imaging. This dual-beam approach has revolutionized TEM sample prep by enabling site-specific, highly precise thinning.

One key advantage is site-specific sampling. Researchers can pinpoint a tiny feature (like a single transistor or a specific grain boundary) and use the ion beam to extract just that area for TEM analysis. This targeted approach is invaluable for studies where knowing the exact origin of the sample is critical. In contrast, older methods involve thinning a broad area and hoping the region of interest lies in the thinnest part.

The FIB-SEM’s precision ensures controlled thickness with minimal damage. Instead of mechanical grinding, which may introduce stress, the ion beam mills a thin lamella with nanometer accuracy. Modern FIB workflows can routinely produce regions below 100 nm thick, and even thin them to ~30 nm for atomic-resolution studies. Throughout milling, SEM imaging monitors progress so the final lamella is exactly on target.

Though FIB milling can cause slight surface damage (ion implantation or amorphization), a final low-kV polish largely removes these effects. Compared to broad-beam ion milling, FIB-SEM offers a more targeted approach with fewer artifacts. In short, it has become the go-to tool for producing high-quality TEM samples and overcoming prep challenges in one platform.

Step-by-Step Workflow

A typical FIB-SEM TEM sample preparation workflow involves these steps:
  • 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

    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

      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 Featured Image

      Understanding Radiation Damage in Cryo-Electron Microscopy

      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-/Ų). 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-/Ų. 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:

      1. 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)

        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:

            1. Savva C. A beginner's guide to cryogenic electron microscopy. Biochem (Lond). 2019;41 (2):46–52. doi:10.1042/BIO04102046.
            2. 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.
            3. Benjin X, Ling L. Developments, applications, and prospects of cryo-electron microscopy. Protein Sci. 2020;29(4):872-882. doi:10.1002/pro.3805.
            4. 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?

            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:
            1. 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.
            2. 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.
            3. 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

              Using Cryo-EM to Determine the Structure of Macromolecular Complexes

              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.


              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

              1. 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.
              2. 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
              3. 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.
              4. 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 Featured Image

                A Walkthrough of the Cryo-EM Workflow

                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

                  What is Transmission Electron Microscopy?

                  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.
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