Electron Tomography of Ice-Embedded Prokaryotic Cells

Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography

Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context. In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

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.

Electron tomography (ET) is an outstanding technique to access information about nanoparticles whose properties are critically dependent on their 3D shape and structure. A particularly relevant application of ET concerns plasmonic chiral nanoparticles, which exhibit strong correlation between optical activity and asymmetric morphology. Thus, it is crucial to go beyond qualitative visual inspection of ET reconstructions and extract quantitative shape or chirality descriptors. However, the time‐consuming nature of conventional ET, where a series of 2D projection images is used for one 3D reconstruction, makes it challenging to collect statistically representative data. A recently proposed semi‐automated approach for the acquisition of the required tilt series can reduce the data collection time from typically ≈ 45 minutes–1 hour to less than 10 minutes. What remains unclear is how this method influences the quality of the reconstruction and the accuracy of the samples’ quantitative descriptors. Here, two sets of chiral gold nanoparticles with wrinkled or twisted morphological chirality are used. It is demonstrated that the fast tilt series acquisitions yield results that are consistent with conventional ET, provided a standardized data processing protocol is used. Finally, a practical example of how fast ET can be used to obtain more statistically relevant measurements is presented.

Approaches to large-scale structures

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.

Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

3D quantitative characterization of metal catalysts supported on heavy oxides by High Angle Annular Dark Field (HAADF) STEM electron tomography (ET) is a very challenging task. Noble metal nanoparticles (Au, Ru) supported on ceria or ceria mixed oxides illustrates the case. The difference, very small in some cases, between the atomic number (Z) of metal and support (Z Ru =44, Z Au =79, Z Ce =58) complicates the discrimination of the small particles. This makes that each step, acquisition, alignment, reconstruction and segmentation, has to be carefully optimized not only to improve the visualization of the nanoparticles but also to extract relevant quantitative results, e.g . metal loading, specific surface area, or particle size distribution. In this work, we have combined advanced image processing algorithms, based on undecimated wavelets transform (UWT) [1], to improve the contrast and denoising the tilt series projections, with new reconstruction algorithms, based on compressed sensing (CS) [2], to study a series of catalysts with a high potential in different processes related to the production of hydrogen, as CeO 2 Nanorods, Au/CeO 2 Nanocubes or Ru/Ce 2 Z 2 O 8 . The results obtained by ET have been compared to those determined by macroscopic characterization techniques as Inductively Coupled Plasma (ICP) or Brunauer, Emmett and Teller (BET) isotherm method. HAADF‐STEM ET has been carried out using a FEI TITAN 3 THEMIS 60‐300 operated at 200kV recently installed at Cadiz University. Data collections were obtained by tilting the specimen about a single axis perpendicular to the electron beam. Series of projections were acquired between ‐70º and +70° either every 2° or 5º. The images series were aligned using Inspect3D and TomoJ and reconstructed using the ASTRA Toolbox implemented in Matlab [3]. In the particular case of CS, Total Variation Minimization (TVM) was carried by using the TVAL3 solver [2]. Figure 1 shows the 3D‐rendered voxel of a catalyst consisting of Au nanoparticles supported on CeO 2 Nanocubes after reconstructing the raw tilt series by SIRT (Figure 1a) and TVM (Figure 1b) and after denoising the tilt series projections by UWT and reconstructed by SIRT (Figure 1c) and TVM (Figure1d). Note how in the case of UWT‐TVM reconstruction an important improvement in terms of morphology of the support and visualization of nanoparticles is obtained. The 3D rendered surface of a similar sample and the 3D quantifications of relevant properties as metal loading, specific surface area and average particle size are shown in Figure 2. It is important to point out how the values are quite similar to those determined by macroscopic characterization techniques. These results indicate that this combination of techniques allows determining nanostructural features representative of the catalysts at macroscopic level.

EPiK-a Workflow for Electron Tomography in Kepler

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

Compressed sensing electron tomography of needle-shaped biological specimens – Potential for improved reconstruction fidelity with reduced dose

Electron tomography (ET) offers a unique capacity to image biological structures in situ. However, the resolution of ET reconstructed tomograms is not comparable to that of the single-particle cryo-EM. If many copies of the object of interest are present in the tomograms, their structures can be reconstructed in the tomogram, picked, aligned and averaged to increase the signal-to-noise ratio and improve the resolution, which is known as the subtomogram averaging. To date, the resolution improvement of the subtomogram averaging is still limited because each reconstructed subtomogram is of low reconstruction quality due to the missing wedge issue. In this article, we propose a novel computational model, the constrained reconstruction model (CRM), to better recover the information from the multiple subtomograms and compensate for the missing wedge issue in each of them. CRM is supposed to produce a refined reconstruction in the final turn of subtomogram averaging after alignment, instead of directly taking the average. We first formulate the averaging method and our CRM as linear systems, and prove that the solution space of CRM is no larger, and in practice much smaller, than that of the averaging method. We then propose a sparse Kaczmarz algorithm to solve the formulated CRM, and further extend the solution to the simultaneous algebraic reconstruction technique (SART). Experimental results demonstrate that CRM can significantly alleviate the missing wedge issue and improve the final reconstruction quality. In addition, our model is robust to the number of images in each tilt series, the tilt range and the noise level. The codes of CRM-SIRT and CRM-SART are available at https://github.com/icthrm/CRM. Supplementary data are available at Bioinformatics online.

We present the top-bottom effect (TBE) of a 5 microm thick amorphous specimen in a 3 MV ultrahigh voltage electron microscope (ultra-HVEM) and its influence on the quality of electron tomography (ET). The 40-nm gold particles on the top surface of the specimen tilted at different angles have been observed to be of poorer image quality than those on the symmetrical bottom surface obtained by tilting and turning over the specimen. The point spread function of the gold-particle images was calculated using the increment Wiener filter and the image-quality variation was then evaluated. The TBE is shown to become more remarkable with the increase of the effective thickness of the tilted specimen or the decrease of the magnification of the ultra-HVEM. The ET simulation indicates that the TBE may generate approximately 4% distortion in the radial direction of a reconstructed sphere model.