Template matching as a tool for annotation of tomograms of stained biological structures

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.     

Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography

Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.     

Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography

The localization of scarce antigens in thin sections of biological material can be accomplished by pre-embedment labeling with ultrasmall immuno-gold labels. Moreover, with this method, labeling is not restricted to the section surface but occurs throughout the section volume. Thus, when combined with electron tomography, antigens can be localized in three dimensions in relation to the 3D (three-dimensional) ultrastructure of the cell. However, for visualization in a transmission electron microscope, these labels need to be enlarged by silver or gold enhancement. The increase in particle size reduces the resolution of the antigen detection and the large particles obscure ultrastructural details in the tomogram. In this paper we show for the first time that these problems can be avoided and that ultrasmall gold labels can be localized in three dimensions without the need for gold or silver enhancement by using HAADF-STEM (high angular annular dark-field-scanning transmission electron microscopy) tomography. This method allowed us to three-dimensionally localize Aurion ultrasmall goat anti-rabbit immuno-gold labels on sections of Epon-embedded, osmium-uranium-lead-stained biological material. Calculations show that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy. We conclude that it is possible to combine the high-fidelity structural information of TEM tomograms with the ultrasmall label localization ability of HAADF-STEM tomograms.     

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.     

A novel three-dimensional variant of the watershed transform for segmentation of electron density maps

Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratios. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. Here, we present a segmentation approach targeting three-dimensional electron density maps derived by electron microscopy. The approach consists of a novel three-dimensional variant of the immersion-based watershed algorithm. We tested the algorithm on calculated data and applied it to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures. The results indicate that the algorithm is reliable, efficient, accurate, and applicable to a wide variety of biological problems.     

Segmentation of electron tomographic data sets using fuzzy set theory principles

In electron tomography the reconstructed density function is typically corrupted by noise and artifacts. Under those conditions, separating the meaningful regions of the reconstructed density function is not trivial. Despite development efforts that specifically target electron tomography manual segmentation continues to be the preferred method. Based on previous good experiences using a segmentation based on fuzzy logic principles (fuzzy segmentation) where the reconstructed density functions also have low signal-to-noise ratio, we applied it to electron tomographic reconstructions. We demonstrate the usefulness of the fuzzy segmentation algorithm evaluating it within the limits of segmenting electron tomograms of selectively stained, plastic embedded spiny dendrites. The results produced by the fuzzy segmentation algorithm within the framework presented are encouraging.     

Bridging the imaging gap: visualizing subcellular architecture with electron tomography

Transmission electron microscopy is a powerful tool that is used to explore the internal structure of tissues, cells, organelles and macromolecular complexes. By integrating data from a series of images in which the orientation of the specimen is progressively varied relative to the incident electron beam it is also possible to extend electron microscopic imaging into the third dimension. This approach, commonly referred to as electron tomography, has been greatly aided in recent years by advances in technology for imaging specimens at cryogenic temperatures, as well as by substantial progress in procedures for automated data collection and image processing. The intense pace of developments in this field is inspired, in a large part, by the hope that the quality of the data will ultimately be good enough to allow interpretation of tomograms of cells, organelles, bacteria and viruses in terms of the three-dimensional spatial arrangements of the constituent molecules.     

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.     

Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes

Electron tomography is a technique for three-dimensional reconstruction, that is widely used for imaging macromolecules, macromolecular assemblies or whole cells. Combined with cryo-electron microscopy, it is capable of visualizing structural detail in a state close to in vivo conditions in the cell. In electron tomography, micrographs are taken while tilting the specimen to different angles about a fixed axis. Due to mechanical constraints, the angular tilt range is limited. As a consequence, the reconstruction of a 3D image is missing data, which for a single axis tilt series is called the “missing wedge”, a region in reciprocal space where Fourier coefficients cannot be obtained experimentally. Tomographic data is analyzed by extracting subvolumes from the raw tomograms, by alignment of the extracted subvolumes, multivariate data analysis, classification, and class-averaging, which results in an increased signal-to-noise ratio and substantial data reduction. Subvolume analysis is a valuable tool to discriminate heterogeneous populations of macromolecules, or conformations of a macromolecule or macromolecular assembly as well as to characterize interactions between macromolecules. However, this analysis is hampered by the lack of data in the original tomograms caused by the missing wedge. Here, we report enhancements of our subvolume processing protocols in which the problem of the missing data in reciprocal space is addressed by using constrained correlation and weighted averaging in reciprocal space. These procedures are applied to the analysis of myosin V and simian immunodeficiency virus (SIV) envelope spikes. We also investigate the effect of the missing wedge on image classification and establish limits of reliability by model calculations with generated phantoms.     

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.     

Mean curvature motion facilitates the segmentation and surface visualization of electron tomograms

Three-dimensional visualization of biological samples is essential for understanding their architecture and function. However, it is often challenging due to the macromolecular crowdedness of the samples and low signal-to-noise ratio of the cryo-electron tomograms. Denoising and segmentation techniques address this challenge by increasing the signal-to-noise ratio and by simplifying the data in images. Here, mean curvature motion is presented as a method that can be applied to segmentation results, created either manually or automatically, to significantly improve both the visual quality and downstream computational handling. Mean curvature motion is a process based on nonlinear anisotropic diffusion that smooths along edges and causes high-curvature features, such as noise, to disappear. In combination with level-set methods for image erosion and dilation, the application of mean curvature motion to electron tomograms and segmentations removes sharp edges or spikes in the visualized surfaces, produces an improved surface quality, and improves overall visualization and interpretation of the three-dimensional images.     

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological “nanomachines” it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 μm in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.     

Assessing the benefits of focal pair cryo-electron tomography

Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic 'focal pairs', which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3 nm for cells thicker than 500 nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

Morphological characterization of molecular complexes present in the synaptic cleft

We obtained tomograms of isolated mammalian excitatory synapses by cryo-electron tomography. This method allows the investigation of biological material in the frozen-hydrated state, without staining, and can therefore provide reliable structural information at the molecular level. We developed an automated procedure for the segmentation of molecular complexes present in the synaptic cleft based on thresholding and connectivity, and calculated several morphological characteristics of these complexes. Extensive lateral connections along the synaptic cleft are shown to form a highly connected structure with a complex topology. Our results are essentially parameter-free, i.e., they do not depend on the choice of certain parameter values (such as threshold). In addition, the results are not sensitive to noise; the same conclusions can be drawn from the analysis of both nondenoised and denoised tomograms.     

The three-dimensional arrangement of intermediate filaments in Romney wool cortical cells

The three-dimensional orientation and arrangement of intermediate filaments in Romney wool ortho-, meso-, and paracortical cells has been revealed using single axis high voltage electron tomography. Modelled tomograms confirm that intermediate filaments in orthocortical cells are arranged helically, with the helical angle progressively increasing from the centre to the periphery of macrofibrils. Intermediate filaments in meso- and paracortical cells display parallel arrangements differing mainly in packing density, with the mesocortex packed more tightly than the paracortex. The intermediate filament arrangements observed confirm expectations based on earlier two-dimensional transmission electron microscopy observations by the authors and other researchers. It is expected that these findings will contribute to a better understanding of the biological and structural basis of wool fibre curvature.     

Electron tomography of biological samples

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1497–1505.Original Russian Text Copyright © 2004 by Marco, Boudier, Messaoudi, Rigaud.     

The discriminative bilateral filter: an enhanced denoising filter for electron microscopy data

Advances in three-dimensional (3D) electron microscopy (EM) and image processing are providing considerable improvements in the resolution of subcellular volumes, macromolecular assemblies and individual proteins. However, the recovery of high-frequency information from biological samples is hindered by specimen sensitivity to beam damage. Low dose electron cryo-microscopy conditions afford reduced beam damage but typically yield images with reduced contrast and low signal-to-noise ratios (SNRs). Here, we describe the properties of a new discriminative bilateral (DBL) filter that is based upon the bilateral filter implementation of Jiang et al. (Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chiu, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struc. Biol. 128, 82-97.). In contrast to the latter, the DBL filter can distinguish between object edges and high-frequency noise pixels through the use of an additional photometric exclusion function. As a result, high frequency noise pixels are smoothed, yet object edge detail is preserved. In the present study, we show that the DBL filter effectively reduces noise in low SNR single particle data as well as cellular tomograms of stained plastic sections. The properties of the DBL filter are discussed in terms of its usefulness for single particle analysis and for pre-processing cellular tomograms ahead of image segmentation.