Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Stain density is an important parameter for optimising the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue and liver tissue.     

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.     

Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200 A the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5 mrad there is insufficient signal at a dose of 5 electrons/A(2) to produce an image with more than 1 electron/per pixel. For larger molecules (>100 A thick which corresponds to 420 kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100 kV. This limit could be extended to about 200 A (about 3 MDa) if a 400 kV microscope were used.     

Computer-controlled spot-scan imaging of crotoxin complex crystals with 400 keV electrons at near-atomic resolution.

400 keV electrons yield a better relative image contrast than 100 keV electrons for a beam-sensitive organic crystal when spot-scan imaging is used [J. Brink and W. Chiu, J. Microscopy 161 (1991) 279]. A FORTRAN 77 program has been written to operate the spot-scan imaging system on a computer workstation under the VMS operating system which is interfaced serially to the JEOL4000 electron microscope. We demonstrate the application of this implementation by imaging crotoxin complex crystals embedded in either vitreous ice or glucose to 2.5 A resolution. The intensity strength of the structure factors of this protein crystal are different at low (> 10 A) resolution but similar at high resolution (< 10 A) for the two embedding media as expected from their scattering contrast difference. Based on our experience as judged from the electron diffraction patterns of highly tilted crystals, flat crystals embedded in glucose can be readily obtained. Furthermore, our spot-scan imaging system also has the option of correcting the focus gradient that is present in images of tilted specimens.     

CTF determination and correction in electron cryotomography

Electron cryotomography (cryoET) has the potential to elucidate the structure of complex biological specimens at molecular resolution but technical and computational improvements are still needed. This work addresses the determination and correction of the contrast transfer function (CTF) of the electron microscope in cryoET. Our approach to CTF detection and defocus determination depends on strip-based periodogram averaging, extended throughout the tilt series to overcome the low contrast conditions found in cryoET. A method for CTF correction that deals with the defocus gradient in images of tilted specimens is also proposed. These approaches to CTF determination and correction have been applied here to several examples of cryoET of pleomorphic specimens and of single particles. CTF correction is essential for improving the resolution, particularly in those studies that combine cryoET with single particle averaging techniques.     

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.     

Instrumental requirements for high resolution imaging

The resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma-free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution-checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on-line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful. Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low-Z specimens, particularly in the dark field mode. Finally, some ‘non-conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.     

High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

A longstanding limitation of imaging with serial block‐face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block‐face due to image jitter. Typically, variable‐pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal‐to‐noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block‐face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block‐face ultramicrotome. This system enables the application of nitrogen gas precisely over the block‐face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high‐resolution block‐face imaging of even the most charge prone of epoxy‐embedded biological samples.     

Quantitative energy-filtered electron microscopy of biological molecules in ice.

The theoretical and experimental bases for quantitative electron microscopy of frozen-hydrated specimens are described, with special considerations of energy filtration to improve the images. The elastic and inelastic scattering from molecules in vacuum and in ice are calculated, and simple methods to approximate scattering are introduced. Multiple scattering calculations are used to describe the scattering from vitreous ice and to predict the characteristics of images of frozen-hydrated molecules as a function of ice thickness and accelerating voltage. Energy filtration is predicted to improve image contrast and signal-to-noise ratio. Experimental values for the inelastic scattering of ice, the energy spectrum of thick ice, and the contrast of biological specimens are determined. The principles of compensation for the contrast transfer function are presented. Tobacco mosaic virus is used to quantify the accuracy of interpreting image intensities to derive the absolute mass, mass per unit length, and internal mass densities of biological molecules. It is shown that compensation for the contrast transfer function is necessary and sufficient to convert the images into accurate representations of molecular density. At a resolution of 2 nm, the radial density reconstructions of tobacco mosaic virus are in quantitative agreement with the atomic model derived from X-ray results.     

Zero-loss energy-filtered imaging of frozen-hydrated proteins: model calculations and implications for future developments

Energy-filtered transmission electron microscopes operating in zero-loss mode are used increasingly to study biological material in frozen-hydrated conditions. The contrast enhancement and improved structural resolution obtainable by this method have been studied using Monte-Carlo model calculations for the scattering processes occurring in such samples. Three models representing typical situations have been analysed, each normalized to minimal beam damage. It is shown that for proteins in thin layers of ice an optimal signal-to-noise ratio is achieved in the 80–120-keV electron energy range. For proteins which have to be embedded in thicker ice layers, a considerably higher acceleration voltage is required. In particular, electron energies above 200 keV would be desirable for electron diffraction work on microcrystals.     

Mechanisms of decoherence in electron microscopy

The understanding and where possible the minimisation of decoherence mechanisms in electron microscopy were first studied in plasmon loss, diffraction contrast images but are of even more acute relevance in high resolution TEM phase contrast imaging and electron holography. With the development of phase retrieval techniques they merit further attention particularly when their effect cannot be eliminated by currently available energy filters. The roles of electronic excitation, thermal diffuse scattering, transition radiation and bremsstrahlung are examined here not only in the specimen but also in the electron optical column. Terahertz-range aloof beam electronic excitation appears to account satisfactorily for recent observations of decoherence in electron holography. An apparent low frequency divergence can emerge for the calculated classical bremsstrahlung event probability but can be ignored for photon wavelengths exceeding the required coherence distance or path lengths in the equipment. Most bremsstrahlung event probabilities are negligibly important except possibly in large-angle bending magnets or mandolin systems. A more reliable procedure for subtracting thermal diffuse scattering from diffraction pattern intensities is proposed.     

Emission microscopy and related techniques: resolution in photoelectron microscopy, low energy electron microscopy and mirror electron microscopy.

A unified treatment of the resolution of three closely related techniques is presented: emission electron microscopy (particularly photoelectron microscopy, PEM), low energy electron microscopy (LEEM), and mirror electron microscopy (MEM). The resolution calculation is based on the intensity distribution in the image plane for an object of finite size rather than for a point source. The calculations take into account the spherical and chromatic aberrations of the accelerating field and of the objective lens. Intensity distributions for a range of energies in the electron beam are obtained by adding the single-energy distributions weighted according to the energy distribution function. The diffraction error is taken into account separately. A working resolution is calculated that includes the practical requirement for a finite exposure time, and hence a finite non-zero current in the image. The expressions for the aberration coefficients are the same in PEM and LEEM. The calculated aberrations in MEM are somewhat smaller than for PEM and LEEM. The resolution of PEM is calculated to be about 50 A, assuming conventional UV excitation sources, which provide current densities at the specimen of 5 x 10(-5) A/cm2 and emission energies ranging up to 0.5 eV. A resolution of about 70 A has been demonstrated experimentally. The emission current density at the specimen is higher in LEEM and MEM because an electron gun is used in place of a UV source. For a current density of 5 x 10(-4) A/cm2 and the same electron optical parameters as for PEM, the resolution is calculated to be 27 A for LEEM and 21 A for MEM.     

Real imaging and size values of Saccharomyces cerevisiae cells with comparable contrast tuning to two environmental scanning electron microscopy modes

The combined application of electron microscopy (EM) is frequently used for the microstructural investigation of biological specimens and plays two important roles in the quantification and in gaining an improved understanding of biological phenomena by making use of the highest resolution capability provided by EM. The possibility of imaging wet specimens in their "native" states in the environmental scanning electron microscope (ESEM) at high resolution and large depth of focus in real time is discussed in this paper. It is demonstrated here that new features can be discovered by the elimination of even the least hazardous approaches in some preparation techniques, that destroy the samples. Since the analysis conditions may influence the morphology and the extreme surface sensitivity of living biological systems, the results obtained from the same cultured cell with two different ESEM modes (Lvac mode and wet mode) were compared. This offers new opportunities compared with ESEM-wet/Lvac-mode imaging, since wet-mode imaging involves a real contrast and gives an indication of the changes in cell morphology and structure required for cell viability. In this study, wet-mode imaging was optimized using the unique ability of cell quantities for microcharacterization in situ giving very fine features of topological effects. Accordingly, the progress is reported by comparing the results of these two modes, which demonstrate interesting application details. In general, the functional comparisons have revealed that the fresh unprocessed Saccharomyces cerevisiae cells (ESEM-wet mode) were essentially unaltered with improved and minimal specimen preparation timescales, and the optimal cell viability degree was visualized and also measured quantitatively while the cell size remained unchanged with continuous images.     

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.     

Iodine vapor staining for atomic number contrast in backscattered electron and X‐ray imaging

Iodine imparts strong contrast to objects imaged with electrons and X‐rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation‐deposition state‐change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas‐tight container along with a small mass of solid I₂. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin‐embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X‐ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron‐ and photon‐sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.     

High resolution dark-field electron microscopy

In the last few years some promising images of biological specimens have been obtained using the high contrast and resolution of dark-field electron microscopy. However, important problems of image interpretation and difficulties in specimen preparation limit at the present time, the usefulness of this mode of image formation. The destruction of the sample by the electron beam, is of utmost importance. Some possibilities of partly overcoming it are discussed.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.