In-situ straining and time-resolved electron tomography data acquisition in a transmission electron microscope.

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins’ functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000–160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.

Janus nanoparticles are nanoparticles (NPs) displaying two sides of different chemical nature which makes them of high interest. The formation of the Janus shell provides the NPs an amphiphilic character, which can therefore self‐assemble in a tunable fashion by varying different experimental parameters such as size, polymer ratio and temperature. Here, Au NPs coated by polyethylene glycol (PEG) and both polystyrene (PS) and poly(N‐isopropylacrylamide) (PNIPAM) were investigated. [1] The true Janus character of the NPs was determined by electron tomography. Characterization of polymer coated particles by electron microscopy is challenging due to the low contrast of the organic materials. The goal was to distinguish the two different polymers in two types of particles (PEG+PNIPAM and PEG+PS coated Au NPs). Different approaches were used to improve the contrast between the two polymers. The PEG+PNIPAM particles were characterized by specific staining of the hemispheres while the character of the PEG+PS particles was investigated by the growth of silica. First, the PEG+PNIPAM coated NPs were stained with 3 mM CuSO 4 .5H 2 O solution to generate sufficient contrast in the Transmission Electron Microscope (TEM). PNIPAM‐coated NPs showed damage of the polymer upon electron beam irradiation. For PEG‐coated NPs a stable shell was observed. In an attempt to distinguish PNIPAM and PEG, PEG+PNIPAM coated particles were stained and investigated with TEM (Fig. 1). Since conventional TEM images are 2D projections from a 3D object, electron tomography was performed. It must be noted that the conventional technique for tomography in materials science, high angle annular dark field scanning TEM (HAADF‐STEM), could not be applied due to the large difference in atomic number Z for Au and the elements in the polymer shell. Instead, Bright‐Field TEM tomography was applied. Images were acquired over the largest possible range (‐76° to +76°) every 2°, aligned and reconstructed by the SIRT (Simultaneous Iterative Reconstruction Technique) algorithm in the Astra Toolbox. [2] The obtained 3D reconstruction displayed rather low contrast between the shell and the background. Manual segmentation based on the orthoslices (xy‐, yx‐ and zx‐direction) could be carried out. This method provided a 3D reconstruction that clearly confirms the formation of stained half‐shells (Fig. 3). In the second case of PEG+PS coated Au NPs, an attempt of staining with calcium phosphate was carried out. In TEM images hemispheres could be observed, but selective staining could not be confirmed. Due to the high affinity of PEG by silica, PEG is able to act as a primer to promote silica condensation. Since PS is hydrophobic and does not allow silica nucleation, the silica shell was expected to occur only at the parts of the NPs that are coated by PEG (Fig. 2). Either staining or silica shell growth over PEG‐coated areas resulted in the observation of semi‐shells (Janus character) in Au NPs by TEM and electron tomography.

Electron tomography is a mature 3D nanocharacterisation technique with applications to almost all classes of materials. It offers 3D resolution at the nanometre level, or better, in volumes of a few hundred nanometres—precisely the playground of the nanoscientist. Electron tomography is especially suited for uncovering the links between structure and properties in systems that show complex 3D character. This chapter offers a comprehensive introduction to the technique, from its mathematical background to practical experimental issues and from tomographic reconstruction to quantitative analysis of 3D volumes. Being angled more towards the physical scientist, this chapter concentrates on the particular challenges of acquiring tomographic data from systems in physical, chemical and materials science using the panoply of imaging techniques available in the modern transmission electron microscope (TEM). These include scanning TEM (STEM), dark-field TEM and chemically sensitive 3D imaging via electron energy loss spectroscopy (EELS) and energy-dispersive X-ray (EDX) spectroscopy. The chapter concludes by examining emerging areas such as atomic-resolution 3D imaging and scanning confocal electron microscopy (SCEM).

Sampling limits for electron tomography with sparsity-exploiting reconstructions

EPiK-a Workflow for Electron Tomography in Kepler

High-Throughput Immunofluorescence and Electron Tomography to Characterize Centrosomal Aberrations in Plasma Cell Neoplasia

Chirality in gold nanostructures offers an exciting opportunity to tune their differential optical response to left- and right-handed circularly polarized light, as well as their interactions with biomolecules and living matter. However, tuning and understanding such interactions demands quantification of the structural features that are responsible for the chiral behavior. Electron tomography (ET) enables structural characterization at the single-particle level and has been used to quantify the helicity of complex chiral nanorods. However, the technique is time-consuming and consequently lacks statistical value. To address this issue, we introduce herein a high-throughput methodology that combines images acquired by secondary electron-based electron beam-induced current (SEEBIC) with quantitative image analysis. As a result, the geometric chirality of hundreds of nanoparticles can be quantified in less than 1 h. When combining the drastic gain in data collection efficiency of SEEBIC with a limited number of ET data sets, a better understanding of how the chiral structure of individual chiral nanoparticles translates into the ensemble chiroptical response can be reached.

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.

In electron tomography (ET) based on a transmission electron microscope, the effective thickness of a specimen increases with the tilt angle and, therefore, the projection quality may deteriorate because of electron scattering. The information-missing region, however, can be reduced by broadening the specimen tilt range. To clarify the general influence of these effects on ET, the projection quality varying with the tilt angle was quantitatively evaluated for a 5-microm thick specimen observed with a 3 MV ultrahigh-voltage electron microscope. Simulations of three-dimensional reconstruction were then performed for different specimen thicknesses and tilt ranges. As a result, the ET accuracy was shown to decrease as the specimen thickness increased. However, an optimum specimen-tilt range, at which ET could reach its highest accuracy, was found to exist and become small with the increase of the specimen thickness. The presented results are helpful for determining the specimen thickness limitation on the ET resolution and improving the ET fidelity of thick specimens.

Primary ciliary dyskinesia with normal ultrastructure: three-dimensional tomography detects absence of DNAH11.

ABSTRACTPrimary Ciliary Dyskinesia (PCD) is a heterogeneous genetic condition characterized by dysfunction of motile cilia. Patients suffer from chronic infection and inflammation of the upper and lower respiratory tract. Diagnosis of PCD is confirmed by identification of a hallmark defect of ciliary ultrastructure or by identification of biallelic pathogenic mutations in a known PCD gene. Since the first description of PCD in 1976, assessment of ciliary ultrastructure by transmission electron microscopy (TEM) has been central to diagnosis and research. Electron tomography is a technique whereby a series of transmission electron micrographs are collected at different angles and reconstructed into a single 3D model of a specimen. Electron tomography provides improved spatial information and resolution compared to a single micrograph. Research by electron tomography has revealed new insight into ciliary ultrastructure and consequently ciliary function at a molecular and cellular level. Gene discovery studies in PCD have utilized electron tomography to define the structural consequences of variants in cilia genes. Modern transmission electron microscopes capable of electron tomography are increasingly being installed in clinical laboratories. This presents the possibility for the use of tomography technique in a diagnostic setting. This review describes the electron tomography technique, the contribution tomography has made to the understanding of basic cilia structure and function and finally the potential of the technique for use in PCD diagnosis.

The rapid progress in materials science that enables the design of materials down to the nanoscale also demands characterization techniques able to analyze the materials down to the same scale, such as transmission electron microscopy. As Belgium’s foremost electron microscopy group, among the largest in the world, EMAT is continuously contributing to the development of TEM techniques, such as high-resolution imaging, diffraction, electron tomography, and spectroscopies, with an emphasis on quantification and reproducibility, as well as employing TEM methodology at the highest level to solve real-world materials science problems. The lab’s recent contributions are presented here together with specific case studies in order to highlight the usefulness of TEM to the advancement of materials science.

This review of the development and current status of electron tomography deals mainly with the mathematical and algorithmic aspects. After a very brief description of the role of electron tomography in structural biology, we turn our attention to the derivation of the forward operator. Starting from the Schrödinger equation, the electron–specimen interaction is modelled as a diffraction tomography problem and the picture is completed by adding a description of the optical system of the transmission electron microscope. The first-order Born approximation enables one to explicitly express the intensity for any finite wavenumber in terms of the propagation operator acting on the specimen convolved with a point spread function, derived from the optics in the transmission electron microscope. Next, we focus on the difficulties that cause the reconstruction problem to be quite challenging. Special emphasis is put on explaining the extremely low signal-to-noise ratio in the data combined with the incomplete data problems, which lead to severe ill-posedness. The next step is to derive the standard phase contrast model used in the electron tomography community. The above-mentioned expression for the intensity generalizes the standard phase contrast model which can be obtained by replacing the propagation operator by its high-energy limit, the x-ray transform, as the wavenumber tends to infinity. The importance of more carefully including the wave nature of the electron–specimen interaction is supported by performing an asymptotic analysis of the intensity as the wavenumber tends to infinity. Next we provide an overview of the various reconstruction methods that have been employed in electron tomography and we conclude by mentioning a number of open problems. Besides providing an introduction to electron tomography written in the ‘language of inverse problems’, the authors hope to raise interest among experts in integral geometry and regularization theory for the mathematical and algorithmic difficulties that are encountered in electron tomography.

Electron tomography (ET) is a method for three-dimensional (3-D) reconstruction of single, transparent objects from a series of images (i.e., a tilt series) recorded with a transmission electron microscope (EM) (Fig. 4.1). The method is related to the procedures used in medical tomography. The 3-D reconstructions are usually computed from the digitized tilt series after a radial weighting scheme has been applied to the Fourier-transformed data. The ET method can be used to reconstruct in 3-D any object that is transparent enough for projection imaging with the transmission EM. This means that specimens of biological origin are usually available for ET 3-D reconstruction, whereas, e.g., colloidal gold particles are not. An ET-calculated 3-D map could be based on projections from objects that have been visualized by EM in several different ways, i.e., from stained or unstained objects, objects visualized at different energy loss levels or objects embedded in different media. The combined analysis of the 3-D structure, imaged in different ways, could thus become very informative. The general applicability also means that it is not restricted to symmetrical or regularly arranged objects, nor to objects with preferred orientations on a support grid. In its present state, the ET method allows reproducible 3-D reconstructions of single molecular objects, with a resolution in the range of 5 nm, of complex cellular specimens. For isolated objects, free of interfering cellular substances, a somewhat higher resolution can be achieved. The ET method covers the intermediate resolution range where there is no other physical technique available to analyze single molecular complexes.

Purpose: Myosin binding protein C is an accessory protein of vertebrate striated muscle thick filaments. Many studies have aimed to elucidate both its structure and function since mutations in the cardiac isoform (cMyBP-C) gene (MYBPC3) are known to be a leading cause of familial hypertrophic cardiomyopathy (HCM) affecting millions of people worldwide. There is strong evidence that phosphorylation of N-terminus M-domain of cMyBP-C is essential for normal cardiac function and that their dephosphorylation is associated with contractile dysfunction and HCM. However, the details of the underlying molecular-level ultrastructure are still poorly understood. The aim of this work is to provide details of the ultrastructure of thick filaments in different states of cMyBP-C phosphorylation. Methods: Two transgenic mouse strains expressing a constitutive phosphorylation or a non-phosphorylatable cMyBP-C have been compared to a homozygous knockout mouse for cMyBP-C and a wild-type (WT) control. We have carried out detailed transmission electron microscopy (TEM) in intact cardiac muscles, and electron tomography (ET) combined with sub-tomographic averaging. Results: TEM shows that cardiomyocyte ultrastructure is relatively well maintained under the total deletion of cMyBP-C from the heart and due to the expression of constitutive phosphorylation of cMyBP-C. However, the constitutive expression of non-PKA-phosphorylatable cMyBP-C leads to both sarcomeric disarray and mitochondria disruption. Longitudinal views of the average 3D reconstructions employing ET illustrate the surface density features of thick filaments. In the WT, two projection masses can be identified originating from cMyBP-C. Both the absence of cMyBP-C and the presence of constitutive non-phosphorylatable cMyBP-C seem to be associated with poorer structural preservation of the thick filaments. Conclusions: This study shows that cMyBP-C constitutive phosphorylation effectively preserves the intracellular arrangement of cardiac myocytes. However, non-PKA-phosphorylatable cMyBP-C leads to a dramatic overall decrease of tissue organization which is much more deleterious than the total deletion of cMyBP-C. This finding provides a structural explanation for cardiac dysfunction in this sample. This study shows that chemically fixed samples can be used for ET to reveal 3D structural information of thick filaments. Results obtained by using this methodology are in good agreement with previous studies using isolated filament approaches. However, in order to obtain higher resolution, advanced methods of sample processing will be required.