Radiation damage in the high resolution electron microscopy of biological materials: a review.

Radiation damage to a biological specimen arises from a variety of interactions between the illuminating electrons and the atoms in it. The relative probabilities of these events, and the amout of energy transferred, can be calculated from basic physical theory. The microscopic damage caused in a particular specimen in given operating conditions is more difficult to predict, but it can be measured by a number of macroscopic indicators, the chief of which are loss of mass and changes in the energy loss spectrum (or electron diffraction, pattern, if any). For most biological material the observed rate of damage is such as to set a limit to the intensity of illumination, the maximum magnification and the minimum size of detail that can be made visible. Several techniques have been devised and tested for reducing the radiation sensitivity of a specimen, of which cooling to a very low temperature and encasing it in an inert medium are the most effective. If the various protective measures act cooperatively, they could increase the effective resolution of sensitive material by an order of magnitude, making possible electron microscopy of the atomic structure of, for instance, the nucleic acid bases and other macromolecules. The prospects for observing living cells at a resolution better than that of the best optical microscopes would remain very small.     

The future of the electron microscope*

The future of electron microscopy lies as much with the conventional as with the newer instruments. The point resolving power of the latter is likely to be pushed to 1 Å, but only after a considerable effort in solving problems of mechanical and electrical stability. Progress in correcting the lens aberrations is even slower. Techniques of specimen preparation and microscope operation are continuously being improved, but will need even greater refinement if proper use is to be made of a 1 Å resolving power, e.g. for identifying bases in a nucleic acid molecule. Extension of the working voltage to 1 MeV and above is increasing the usefulness of the conventional electron microscope, particularly in metallurgy, and plans are now being made for even higher voltages. Of the newer, unconventional instruments, the scanning electron microscope is already establishing a place for itself, especially in industrial applications where surface conditions on the microscale are important. It is likely to find increasing use in micro-circuitry, but also in some branches of biological research, even if its resolving power cannot be brought below 100 Å. The combination of X-ray spectrometry with electron microscopy holds promise of wide application, in the form of a hybrid electron microscope-microanalyser. Secondary-emission microscopes are slowly finding a place for themselves, mainly in applied science and technology. Mirror microscopes, for surface investigations, and ion microscopes, for microanalysis, are still in an early stage but have interesting possibilities. The field ion microscope, simplest in principle but sophisticated in technique, is unique in showing the position of individual atoms in a metal tip. It is already finding applications, especially in studies of radiation damage.     

Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review.

When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.     

Direct electron imaging in electron microscopy with monolithic active pixel sensors

A new imaging device for dynamic electron microscopy is in great demand. The detector should provide the experimenter with images having sufficient spatial resolution at high speed. Immunity to radiation damage, accumulated during exposures, is critical. Photographic film, a traditional medium, is not adequate for studies that require large volumes of data or rapid recording and charge coupled device (CCD) cameras have limited resolution, due to phosphor screen coupling. CCD chips are not suitable for direct recording due to their extreme sensitivity to radiation damage. This paper discusses characterization of monolithic active pixel sensors (MAPS) in a scanning electron microscope (SEM) as well as in a transmission electron microscope (TEM). The tested devices were two versions of the MIMOSA V (MV) chip. This 1M pixel device features pixel size of 17 x 17 microm(2) and was designed in a 0.6 microm CMOS process. The active layer for detection is a thin (less than 20 microm) epitaxial layer, limiting the broadening of the electron beam. The first version of the detector was a standard imager with electronics, passivation and interconnection layers on top of the active region; the second one was bottom-thinned, reaching the epitaxial layer from the bottom. The electron energies used range from a few keV to 30 keV for SEM and from 40 to 400 keV for TEM. Deterioration of the image resolution due to backscattering was quantified for different energies and both detector versions.     

A simple liquid-He-cooled specimen stage for an ultra-high resolution transmission electron microscope

A simple top-entry specimen holder for an ultra-high resolution transmission electron microscope which has demonstrated 2.5 Å point-to-point resolution at 30 K is described. The stage has proved useful in studying low-temperature solid phases and phase transformations and is expected to be effective in reducing radiation damage for some organic specimens.     

High-resolution scanning electron microscopy.

The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal. Any practical consideration of the high-resolution performance of the SEM must therefore also involve a knowledge of the contrast available from the signal producing the image and the radiation sensitivity of the specimen. With state-of-the-art electron optics, resolutions of the order of 1 nm are now possible. The optimum conditions for achieving such performance with the minimum radiation damage to the specimen correspond to beam energies in the range 1-3 keV. Progress beyond this level may be restricted by the delocalization of SE production and ultimate limits to electron-optical performance.     

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V₀, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V₀=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V₀ to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed.     

Assessment of electron irradiation damage to biomolecules using the Patterson function

A method for the assessment of electron radiation damage to 5-iodouracil is described which involves the use of the Patterson function. Changes in Patterson maps computed from the electron diffraction patterns recorded at increasing electron irradiation have been related to structural and chemical damage to 5-iodouracil. The process of radiation damage is also discussed in terms of a disorder parameter which was found to increase with increasing irradiation.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Johns Hopkins University, Baltimore, Maryland

The STEM can be used in one of three modes: 1) to image individual atoms; 2) to measure mass or molecular weight; 3) to collect electron energy loss spectra or x-ray fluorescence data. Heavy atom imaging is used to identify chemical groups in a molecule or macromolecules in an assembly. Specific labels have been developed for bases in nucleic acids. These permit localization of bound proteins on single strand nucleic acids. Pt(gly-L-met)Cl is a specific label for methionine residues of proteins as shown with the SLS aggregate of collagen. Lysine can be labeled as well if first methyl (methyl-thio-acetimidate) is coupled. This labeling procedure permits the localization of individual histones within a nucleosome. Mass determination can be used to answer crucial questions about biological assemblies. This is demonstrated by examples from muscle structure.     

Quantitative nanoscale water mapping in frozen-hydrated skin by low-loss electron energy-loss spectroscopy

Spatially resolved low-loss electron energy-loss spectroscopy (EELS) is a powerful method to quantitatively determine the water distribution in frozen-hydrated biological materials at high spatial resolution. However, hydrated tissue, particularly its hydrophilic protein-rich component, is very sensitive to electron radiation. This sensitivity has traditionally limited the achievable spatial resolution because of the relatively high noise associated with low-dose data acquisition. We show that the damage caused by high-dose data acquisition affects the accuracy of a multiple-least-squares (MLS) compositional analysis because of inaccuracies in the reference spectrum used to represent the protein. Higher spatial resolution combined with more accurate compositional analysis can be achieved if a reference spectrum is used that better represents the electron-beam-damaged protein component under frozen-hydrated conditions rather than one separately collected from dry protein under low-dose conditions. We thus introduce a method to extract the best-fitting protein reference spectrum from an experimental spectrum dataset. This method can be used when the MLS-fitting problem is sufficiently constrained so that the only unknown is the reference spectrum for the protein component. We apply this approach to map the distribution of water in cryo-sections obtained from frozen-hydrated tissue of porcine skin. The raw spectral data were collected at doses up to 10⁵ e/nm² despite the fact that observable damage begins at doses as low as 10³ e/nm². The resulting spatial resolution of 10 nm is 5–10 times better than that in previous studies of frozen-hydrated tissue and is sufficient to resolve sub-cellular water fluctuations as well as the inter-cellular lipid-rich regions of skin where water-mediated processes are believed to play a significant role in the phenotype of keratinocytes in the stratum corneum.     

Limits to the spatial, energy and momentum resolution of electron energy-loss spectroscopy

We discuss various factors that determine the performance of electron energy-loss spectroscopy (EELS) and energy-filtered (EFTEM) imaging in a transmission electron microscope. Some of these factors are instrumental and have undergone substantial improvement in recent years, including the development of electron monochromators and aberration correctors. Others, such as radiation damage, delocalization of inelastic scattering and beam broadening in the specimen, derive from basic physics and are likely to remain as limitations. To aid the experimentalist, analytical expressions are given for beam broadening, delocalization length, energy broadening due to core-hole and excited-electron lifetimes, and for the momentum resolution in angle-resolved EELS.     

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.     

Discrimination of protein and nucleic acids by electron microscopy using contrast variation

A contrast variation method is described by which protein and nucleic acid components of nucleoprotein assemblies can be distinguished in the electron microscope. Using contrasting media of variable electron scattering density, the contribution to image contrast of protein or nucleic acid can be matched out. The protein and DNA components of T4 bacteriophages were visualized separately in this way. The distribution of RNA and protein in ribosomes was studied by processing images of crystalline sheets of ribosomes embedded in a range of contrasting media. Projection maps of crystalline ribosomes suggest that the ribosomal RNA is located predominantly in the central region of the particle.     

Measurements of the profile of an intense electron beam

Methods for measuring the profiles of high-power electron beams by using a thin tungsten wire moved transversely to the beam have been developed. In one method, the electron current intercepted by the wire is measured and the beam profile is determined from a solution to the Abel equation under the assumption of axial beam symmetry. The second method is based on the detection of the local radiation emitted by the wire being heated by the beam to 1700–2200 K. The wire is additionally heated by an electric current in order to improve the sensitivity and spatial resolution. The measured beam current density is ～5–50 A/cm², and the resolution is ～0.1 cm.     

The extension of scanning transmission electron microscopy by use of diffraction information.

The information contained in the intensity distribution of the convergent beam electron diffraction pattern, produced in the detector plane for each incident beam position in a scanning transmission electron microscope, may be used to enhance the resolution of the microscope or else to decrease the electron irradiation of the specimen involved in deducing information at any particular level of resolution. The structural information concerning the specimen may be obtained, for example by interpretation of the Patterson function using image-seeking methods. The statistical error involved due to the finite number of electrons available may be derived by considering the efficiency of use of the information contained in Patterson function peaks. The most efficient means for using the available information appears to be that based on information theory concepts, which employ the integral over the product of the observed diffraction pattern intensity and the intensity calculated for known or postulated groupings of atoms. The reduction in radiation damage possible by use of this method, relative to that involved in the separate imaging of individual atoms, may be by a factor of approximately n, where n is the number of atoms in the known or postulated group being sought.     

Temperature dependence of the critical electron exposure for hydrocarbon monolayers

Effects of electron-beam-induced radiation damage in monolayers of several saturated fatty acids and related molecules have been measured by observation of the fading of electron diffraction patterns produced by the crystalline monolayers. Measurements were made with the specimens at temperatures in the range from about 6 to 310 K. Near room temperature, exposures of approximately 50 and 200 electrons/nm² are required to reduce the intensities of the diffraction patterns of stearic and behenic acids, respectively, to 1/e of their initial intensities. These critical exposures increase smoothly as the temperature decreases, to 1600 and 2200 e/nm², respectively, at a specimen temperature of about 6 K. A strong correlation was observed between the critical exposures and melting temperatures of the various specimens examined. These results can be interpreted in terms of radiolysis pathways that are highly specific to hydrocarbons.     

Free-radical probes of condensed-phase aspects of radiation damage mechanisms in DNA constituents: 5-halouracils

Employing free radicals as structural and quantitative monitors for radiation-induced alterations, some aspects of condensed- phase damage mechanisms in organic material are discussed using DNA and its constituents as target example. Specifically, reactions in low-temperature glasses are compared with those in aqueous solutions exemplified for the natural nucleic acid constituents uracil and thymine and their 5-halogen substituted derivatives which modify the radiation response. In addition, the topic of energy transfer in solid-state radiation chemistry is outlined and discussed emphasizing again the group of 5-halouracils. A difference in transfer mechanisms between aquated and dry solid systems is outlined and the role of structural water is tentatively implied. The findings are discussed in the context of electron microscopy at low temperatures.     

Probing nuclear ultrastructure by electron spectroscopic imaging

Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20–30 nm. This complexity hinders the study of structure-function relationships within the mammalian nucleus. Element-specific mapping using the energy-filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of stains relative to unstained material when biochemical-specific staining protocols, such as EDTA-regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogeneous. ESI shows substantial promise for the investigation of structure–function relationships in biological specimens.     

The role of electron capture dissociation in biomolecular analysis

The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural analysis of biomolecules. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision-induced dissociation (SORI-CID), are discussed. The instrumental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligonucleotides is also discussed.