Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

Reprint of "Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)" [J. Struct. Biol. 160 (2007) 35-48

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L_2,3 edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell’s nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.     

Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure

Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H₂O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.     

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.     

Image reconstruction using electron micrographs of insect flight muscle. Use of thick transverse sections to supplement data from tilted thin longitudinal sections.

Three-dimensional reconstruction using electron micrographs of thin sections is a powerful technique for determining cross-bridge structure. Tilt restrictions in the electron microscope prevent data collection beyond tilt angles of 60 degrees, giving rise to a "missing cone" of transform data. We show here how much of this data can be obtained using micrographs of thick transverse sections, and the effect this data has on reconstructed images of the insect flight muscle MYAC layer. As a byproduct, the analysis showed that section thinning resulting from prolonged electron irradiation had occurred in the thin longitudinal section used for the previously published MYAC layer reconstruction (Taylor et al., 1984). Comparison of projection density maps calculated from the thin longitudinal section reconstruction and the thick section data show that the data within the missing cone that is not accessible by tilting sharpens the boundaries of the components, flattens the density profile across the thick filament, and enlarges the molecular envelope of the thin filament. We conclude that the reconstructed images of the MYAC layer provide a picture of the structural principles underlying the system but that transform data within the missing cone are necessary to describe accurately the envelopes and profiles of these structural elements.     

Dual-Axis Tomography: An Approach with Alignment Methods That Preserve Resolution

Tomographic reconstructions of biological specimens are now routinely being generated in our high voltage electron microscope by tilting the specimen around two orthogonal axes. Separate tomograms are computed from each tilt series. The two tomograms are aligned to each other with general 3-D linear transformations that can correct for distortions between the two tomograms, thus preserving the inherent resolution of the reconstruction throughout its volume. The 3-D Fourier transforms of the two tomograms are then selectively combined to achieve a single tomogram. Unlike a single-axis tomogram, a dual-axis tomogram shows good resolution for extended features at any orientation in the plane of the specimen; it also has improved resolution in the depth of the specimen. Calculations indicate that the improvements available from double tilting and from tilting to higher angles are largely additive. Actual and model data were used to assess whether varying the increment between tilted views in proportion to the cosine of the tilt angle would allow a reduction in the number of pictures required to achieve a given resolution of reconstruction. Analysis by Fourier sector correlation indicated that the variable tilt increment improved the reconstruction in some respects but degraded it in others. A varying tilt increment thus does not give an unqualified improvement, at least when using back-projection algorithms for the reconstruction.     

Three-dimensional reconstruction using an adaptive simultaneous algebraic reconstruction technique in electron tomography

Three-dimensional (3D) reconstruction of electron tomography (ET) has emerged as an important technique in analyzing structures of complex biological samples. However most of existing reconstruction methods are not suitable for extremely noisy and incomplete data conditions. We present an adaptive simultaneous algebraic reconstruction technique (ASART) in which a modified multilevel access scheme and an adaptive relaxation parameter adjustment method are developed to improve the quality of the reconstructed 3D structure. The reconstruction process is facilitated by using a column-sum substitution approach. This modified multilevel access scheme is adopted to arrange the order of projections so as to minimize the correlations between consecutive views within a limited angle range. In the adaptive relaxation parameter adjustment method, not only the weight matrix (as in the existing methods) but the gray levels of the pixels are employed to adjust the relaxation parameters so that the quality of the reconstruction is improved while the convergence process of the reconstruction is accelerated. In the column-sum substitution approach, the computation to obtain the reciprocal of the sum for the columns in each view is avoided so that the needed computations for each iteration can be reduced. Experimental results show that the proposed technique ASART is better based on objective quality measures than other methods, especially when data is noisy and limited in tilt angles. At the same time, the reconstruction by ASART outperforms that of simultaneous algebraic reconstruction technique (SART) in speed.     

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.     

Studies on the morphopoiesis of the head of phage T-even

Summary The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T₄ amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.     

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 Å. A 3 Å projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as β-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.     

Improved specimen reconstruction by Hilbert phase contrast tomography

The low signal-to-noise ratio (SNR) in images of unstained specimens recorded with conventional defocus phase contrast makes it difficult to interpret 3D volumes obtained by electron tomography (ET). The high defocus applied for conventional tilt series generates some phase contrast but leads to an incomplete transfer of object information. For tomography of biological weak-phase objects, optimal image contrast and subsequently an optimized SNR are essential for the reconstruction of details such as macromolecular assemblies at molecular resolution. The problem of low contrast can be partially solved by applying a Hilbert phase plate positioned in the back focal plane (BFP) of the objective lens while recording images in Gaussian focus. Images recorded with the Hilbert phase plate provide optimized positive phase contrast at low spatial frequencies, and the contrast transfer in principle extends to the information limit of the microscope. The antisymmetric Hilbert phase contrast (HPC) can be numerically converted into isotropic contrast, which is equivalent to the contrast obtained by a Zernike phase plate. Thus, in-focus HPC provides optimal structure factor information without limiting effects of the transfer function. In this article, we present the first electron tomograms of biological specimens reconstructed from Hilbert phase plate image series. We outline the technical implementation of the phase plate and demonstrate that the technique is routinely applicable for tomography. A comparison between conventional defocus tomograms and in-focus HPC volumes shows an enhanced SNR and an improved specimen visibility for in-focus Hilbert tomography.     

Compensation of Missing Wedge Effects with Sequential Statistical Reconstruction in Electron Tomography

Electron tomography (ET) of biological samples is used to study the organization and the structure of the whole cell and subcellular complexes in great detail. However, projections cannot be acquired over full tilt angle range with biological samples in electron microscopy. ET image reconstruction can be considered an ill-posed problem because of this missing information. This results in artifacts, seen as the loss of three-dimensional (3D) resolution in the reconstructed images. The goal of this study was to achieve isotropic resolution with a statistical reconstruction method, sequential maximum a posteriori expectation maximization (sMAP-EM), using no prior morphological knowledge about the specimen. The missing wedge effects on sMAP-EM were examined with a synthetic cell phantom to assess the effects of noise. An experimental dataset of a multivesicular body was evaluated with a number of gold particles. An ellipsoid fitting based method was developed to realize the quantitative measures elongation and contrast in an automated, objective, and reliable way. The method statistically evaluates the sub-volumes containing gold particles randomly located in various parts of the whole volume, thus giving information about the robustness of the volume reconstruction. The quantitative results were also compared with reconstructions made with widely-used weighted backprojection and simultaneous iterative reconstruction technique methods. The results showed that the proposed sMAP-EM method significantly suppresses the effects of the missing information producing isotropic resolution. Furthermore, this method improves the contrast ratio, enhancing the applicability of further automatic and semi-automatic analysis. These improvements in ET reconstruction by sMAP-EM enable analysis of subcellular structures with higher three-dimensional resolution and contrast than conventional methods.     

Local refinement: an attempt to correct for shrinkage and distortion in electron tomography

A critical problem in electron tomography is the deformation of the specimen due to radiation, or "shrinkage," which interferes with image alignment and thereby limits resolution. Here, we describe a general strategy for refining preliminary reconstructions which allows the damage due to the shrinkage of plastic-embedded thin sectioned specimens (50-80 nm) to be corrected. The basic steps of the strategy involve: (a) the partition of the preliminary reconstruction into sub-volumes; (b) the extraction of corresponding sub-areas for each sub-volume from the micrographs of the tilt series; (c) the re-projection of each sub-volume according to the orientation parameters; and (d) the refinement of these parameters by correlating each sub-area to the corresponding computed projection. We tested the strategy by refining chemical synapses reconstructed from series imaged with conical, double and single tilt geometries. The results gathered with local refinement were evaluated by visually inspecting the structure of biological membranes in the maps. In an effort to quantify these improvements, we studied the refined maps using correlation criteria and mapped the corrections applied to the orientation parameters in each sub-volume of the reconstruction. Simulation experiments complemented the data gathered by correlation analysis. Based on these criteria, we concluded that local refinement significantly improves the overall quality of the reconstructions of chemical synapses calculated from series imaged with conical and double tilt geometries.     

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.     

Structure of gel phase DMPC determined by X-ray diffraction

The structure of fully hydrated gel phase dimyristoylphosphatidylcholine lipid bilayers was obtained at 10 degrees C. Oriented lipid multilayers were used to obtain high signal-to-noise intensity data. The chain tilt angle and an estimate of the methylene electron density were obtained from wide angle reflections. The chain tilt angle is measured to be 32.3 +/- 0.6 degrees near full hydration, and it does not change as the sample is mildly dehydrated from a repeat spacing of D = 59.9 A to D = 56.5 A. Low angle diffraction peaks were obtained up to the tenth order for 17 samples with variable D and prepared by three different methods with different geometries. In addition to the usual Fourier reconstructions of the electron density profiles, model electron density profiles were fit to all the low angle data simultaneously while constraining the model to include the wide-angle data and the measured lipid volume. Results are obtained for area/lipid (A = 47.2 +/- 0.5 A(2)), the compressibility modulus (K(A) = 500 +/- 100 dyn/cm), various thicknesses, such as the hydrocarbon thickness (2D(C) = 30.3 +/- 0.2 A), and the head-to-head spacing (D(HH) = 40.1 +/- 0.1 A).     

Automated Electron Microscope Tomography of Frozen–Hydrated Chromatin: The Irregular Three-Dimensional Zigzag Architecture Persists in Compact, Isolated Fibers

The potential of electron microscope tomography as a tool for obtaining three-dimensional (3D) information about large macromolecular assemblies is greatly extended by automation of data collection. With the implementation of automated control of tilting, focusing, and digital image recording described here, tilt series of frozen–hydrated specimens can be collected with the requisite low dose. Long chromatin fibers were prepared in 90 mM monovalent ions to maintain a fully compact conformation, and after vitrification were completely contained within the ice layer. Tilt series of this material were recorded at 5° tilt increments between +60° and −60°, with a cumulative dose of ≈35 e⁻/Ųfor the series. This extremely low dose data was successfully aligned, then reconstructed by weighted backprojection. The underlying architecture of the fibers is an irregular 3D zigzag of interconnected nucleosomes, with the linker DNA between successive nucleosomes in a largely extended conformation. The visualization of this structural motif within long, frozen–hydrated chromatin fibers at relatively high salt extends our previous studies on small fragments at low ionic strength and is in agreement with the observation of this architecture in chromatin fibersin situin sectioned nuclei.     

Lipid bilayer ultrastructure: Electron density profiles and chain tilt angles as determined by X-ray diffraction

High resolution (6 Å) electron density profiles have been computed on an absolute electron density scale for bilayers composed of both saturated fatty acids and fatty acids associated with the alkaline earth series of divalent cations. Low-angle X-ray diffraction data have been interpreted by an isomorphous replacement technique. The position on the X-ray film of discrete wide-angle reflections has provided direct information on the hydrocarbon chain packing and chain tilt in these bilayers. These results have been correlated to an electron microscopy study of the same bilayers (Waldbilling, R. C., Robertson J. D. and McIntosh, T.J. (1976) Biochim. Biophys. Acta 448, 1–14) and also to X-ray diffraction studies of fatty acid crystals. A method for forming and structurally analyzing bilayers of well defined chemical asymmetry is also described.