Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.     

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation.     

TOM software toolbox: acquisition and analysis for electron tomography

Automated data acquisition procedures have changed the perspectives of electron tomography (ET) in a profound manner. Elaborate data acquisition schemes with autotuning functions minimize exposure of the specimen to the electron beam and sophisticated image analysis routines retrieve a maximum of information from noisy data sets. "TOM software toolbox" integrates established algorithms and new concepts tailored to the special needs of low dose ET. It provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis. Designed as a collection of computational procedures it is a complete software solution within a highly flexible framework. TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications.     

Limiting factors in atomic resolution cryo electron microscopy: no simple tricks

To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors--in both cryoEM image acquisition and 3D reconstruction--that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved 'characteristic surfaces' in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.     

High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1–2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.     

Transform-based backprojection for volume reconstruction of large format electron microscope tilt series

Alignment of the individual images of a tilt series is a critical step in obtaining high-quality electron microscope reconstructions. We report on general methods for producing good alignments, and utilizing the alignment data in subsequent reconstruction steps. Our alignment techniques utilize bundle adjustment. Bundle adjustment is the simultaneous calculation of the position of distinguished markers in the object space and the transforms of these markers to their positions in the observed images, along the bundle of particle trajectories along which the object is projected to each EM image. Bundle adjustment techniques are general enough to encompass the computation of linear, projective or nonlinear transforms for backprojection, and can compensate for curvilinear trajectories through the object, sample warping, and optical aberration. We will also report on new reconstruction codes and describe our results using these codes.     

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored. At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible. The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages. When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam. Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell. The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography. It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV). Randomized author sequence.     

The effect of overabundant projection directions on 3D reconstruction algorithms

The experimental process of collecting images from macromolecules in an electron microscope is such that it does not allow for prior specification of the angular distribution of the projection images. As a consequence, an uneven distribution of projection directions may occur. Concerns have been raised recently about the behavior of 3D reconstruction algorithms for the case of unevenly distributed projections. It has been illustrated on experimental data that in the case of a heavily uneven distribution of projection directions some algorithms tend to elongate the reconstructed volumes along the overloaded direction so much as to make a quantitative biological analysis impossible. In answer to these concerns we have developed a strategy for quantitative comparison and optimization of 3D reconstruction algorithms. We apply this strategy to quantitatively analyze algebraic reconstruction techniques (ART) with blobs, simultaneous iterative reconstruction techniques (SIRT) with voxels, and weighted backprojection (WBP). We show that the elongation artifacts that had been previously reported can be strongly reduced. With our specific choices for the free parameters of the three algorithms, WBP reconstructions tend to be inferior to those obtained with either SIRT or ART and the results obtained with ART are comparable to those with SIRT, but at a very small fraction of the computational cost of SIRT.     

Interpretation of electron micrographs of single and serial sections

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

Fast rotational matching of single-particle images

The presence of noise and absence of contrast in electron micrographs lead to a reduced resolution of the final 3D reconstruction, due to the inherent limitations of single-particle image alignment. The fast rotational matching (FRM) algorithm was introduced recently for an accurate alignment of 2D images under such challenging conditions. Here, we implemented this algorithm for the first time in a standard 3D reconstruction package used in electron microscopy. This allowed us to carry out exhaustive tests of the robustness and reliability in iterative orientation determination, classification, and 3D reconstruction on simulated and experimental image data. A classification test on GroEL chaperonin images demonstrates that FRM assigns up to 13% more images to their correct reference orientation, compared to the classical self-correlation function method. Moreover, at sub-nanometer resolution, GroEL and rice dwarf virus reconstructions exhibit a remarkable resolution gain of 10-20% that is attributed to the novel image alignment kernel.     

Focussed ion beam milling and scanning electron microscopy of brain tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.     

Multiple sequence alignment using partial order graphs

MOTIVATION: Progressive Multiple Sequence Alignment (MSA) methods depend on reducing an MSA to a linear profile for each alignment step. However, this leads to loss of information needed for accurate alignment, and gap scoring artifacts. RESULTS: We present a graph representation of an MSA that can itself be aligned directly by pairwise dynamic programming, eliminating the need to reduce the MSA to a profile. This enables our algorithm (Partial Order Alignment (POA)) to guarantee that the optimal alignment of each new sequence versus each sequence in the MSA will be considered. Moreover, this algorithm introduces a new edit operator, homologous recombination, important for multidomain sequences. The algorithm has improved speed (linear time complexity) over existing MSA algorithms, enabling construction of massive and complex alignments (e.g. an alignment of 5000 sequences in 4 h on a Pentium II). We demonstrate the utility of this algorithm on a family of multidomain SH2 proteins, and on EST assemblies containing alternative splicing and polymorphism. AVAILABILITY: The partial order alignment program POA is available at http://www.bioinformatics.ucla.edu/poa.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy

A computational procedure is described for assigning the absolute hand of the structure of a protein or assembly determined by single-particle electron microscopy. The procedure requires a pair of micrographs of the same particle field recorded at two tilt angles of a single tilt-axis specimen holder together with the three-dimensional map whose hand is being determined. For orientations determined from particles on one micrograph using the map, the agreement (average phase residual) between particle images on the second micrograph and map projections is determined for all possible choices of tilt angle and axis. Whether the agreement is better at the known tilt angle and axis of the microscope or its inverse indicates whether the map is of correct or incorrect hand. An increased discrimination of correct from incorrect hand (free hand difference), as well as accurate identification of the known values for the tilt angle and axis, can be used as targets for rapidly optimizing the search or refinement procedures used to determine particle orientations. Optimized refinement reduces the tendency for the model to match noise in a single image, thus improving the accuracy of the orientation determination and therefore the quality of the resulting map. The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy. Structure factor amplitudes of a three-dimensional map of the E2 catalytic core obtained by averaging untilted images of 3667 icosahedral particles are compared to a scattering reference using a Guinier plot. A noise-dependent structure factor weight is derived and used in conjunction with a temperature factor (B=-1000A(2)) to restore high-resolution contrast without amplifying noise and to visualize molecular features to 8.7A resolution, according to a new objective criterion for resolution assessment proposed here.     

A "Long Indel" model for evolutionary sequence alignment

We present a new probabilistic model of sequence evolution, allowing indels of arbitrary length, and give sequence alignment algorithms for our model. Previously implemented evolutionary models have allowed (at most) single-residue indels or have introduced artifacts such as the existence of indivisible "fragments." We compare our algorithm to these previous methods by applying it to the structural homology dataset HOMSTRAD, evaluating the accuracy of (1) alignments and (2) evolutionary time estimates. With our method, it is possible (for the first time) to integrate probabilistic sequence alignment, with reliability indicators and arbitrary gap penalties, in the same framework as phylogenetic reconstruction. Our alignment algorithm requires that we evaluate the likelihood of any specific path of mutation events in a continuous-time Markov model, with the event times integrated out. To this effect, we introduce a "trajectory likelihood" algorithm (Appendix A). We anticipate that this algorithm will be useful in more general contexts, such as Markov Chain Monte Carlo simulations.     

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.     

INTERPRETATIONS OF ELECTRON MICROGRAPHS OF SINGLE AND SERIAL SECTIONS

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

Long-distance interactions between Ehrlich ascites tumour cells.

In previous work, electron micrographs were made of adjacent surfaces of aldehyde-fixed, Ehrlich ascites tumour cells cultured on coverslips, after reacting some of their negatively charged surface sites with colloidal iron hydroxide (CIH) particles. It was observed that microvilli from one cell were aligned with intermicrovillus regions on another, where the density of the adsorbed CIH particles was significantly lower than in adjacent regions. Alignment, which was considered to represent interactions between the two peripheral cellular regions, took place when these regions were apparently separated by more than 200 nm, in an environment of physiologic ionic strength ( 0·145 m NaCl).In this communication we attempt to find feasible mechanisms for the alignment phenomenon in physical terms, in cases where the observed separation of 200 nm is correct, and in cases where the distances are overestimated due to preparative artifacts.It is concluded, that at distances of separation in excess of 200 nm, one feasible mechanism for alignment is that net negatively charged macromolecules diffusing out of cells in the region of their microvilli, electrostatically repel CIH-binding anionic sites in the lipid-rich “fluid” matrix of the periphery of the opposed cell, causing gaps in their distribution. The role of electrostatic and electrodynamic (van der Waals') forces in causing alignment is also discussed in terms of distance of separation.This communication is concerned with the interpretation in terms of various interactions, of electron micrographs showing evidence of alignment between microvilli from one cell with specific areas of another.