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 3Šdetails 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.
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.