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Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer-Lambert law
Abstract
Cryo-electron tomography (cryo-ET) is an emerging technique that can elucidate the architecture of macromolecular complexes and cellular ultrastructure in a near-native state. Some important sample parameters, such as thickness and tilt, are needed for 3-D reconstruction. However, these parameters can currently only be determined using trial 3-D reconstructions. Accurate electron mean free path plays a significant role in modeling image formation process essential for simulation of electron microscopy images and model-based iterative 3-D reconstruction methods; however, their values are voltage and sample dependent and have only been experimentally measured for a limited number of sample conditions. Here, we report a computational method, tomoThickness, based on the Beer-Lambert law, to simultaneously determine the sample thickness, tilt and electron inelastic mean free path by solving an overdetermined nonlinear least square optimization problem utilizing the strong constraints of tilt relationships. The method has been extensively tested with both stained and cryo datasets. The fitted electron mean free paths are consistent with reported experimental measurements. The accurate thickness estimation eliminates the need for a generous assignment of Z-dimension size of the tomogram. Interestingly, we have also found that nearly all samples are a few degrees tilted relative to the electron beam. Compensation of the intrinsic sample tilt can result in horizontal structure and reduced Z-dimension of tomograms. Our fast, pre-reconstruction method can thus provide important sample parameters that can help improve performance of tomographic reconstruction of a wide range of samples.
... Quantitative estimation of ice thickness therefore would be necessary. We have adopted a simple ice-thickness measurement method based on the Beer-Lambert law (Yan et al., 2015) considering its convenience and online measurement. In brief, the ice thickness (t) can be approximated from the ratio of image intensity of a melted area (I 0 ) and an area with ice (I), and the inelastic mean free path of electrons in vitreous ice (l in ) using the equation t = l in ln I 0 I (Figures S1C and S1D). ...
... To minimize the reduction of image contrast by the antibody layer, we have carefully optimized the freezing condition (e.g., blotting time, humidity) for minimal ice thickness. A Beer-Lambert law-based ice-thickness estimation approach (Yan et al., 2015) has been applied during sample screening to quantitatively evaluate the potential for further ice minimization. As such ice-thickness estimation with the Beer-Lambert law-based method can be easily performed by melting a small region of the grid (similar to the sacrificed area for focusing during low-dose imaging), taking an image and simple calculation (Figures S1C and S1D), it provides a convenient and quick feedback for freezing optimization. ...
... Cryo-EM grids of frozen sample under different conditions were checked using a Philips CM200 microscope with a 1 K CCD camera. The overall quality of the frozen grids was first visually evaluated as usual (Grassucci et al., 2007); For promising conditions, the ice thickness was quantitatively estimated using the thickness estimation method based on the Beer-Lambert law (Malis et al., 1988;Yan et al., 2015). More specifically, after melting the ice at a corner of the vitreous ice film, a low-magnification image (e.g., 3,8003) was taken covering both the region of interest and the melted area using a CCD camera. ...
The affinity cryoelectron microscopy (cryo-EM) approach has been explored in recent years to simplify and/or improve the sample preparation for cryo-EM, which can bring previously challenging specimens such as those of low abundance and/or unpurified ones within reach of the cryo-EM technique. Despite the demonstrated successes for solving structures to low to intermediate resolutions, the lack of near-atomic structures using this approach has led to a common perception of affinity cryo-EM as a niche technique incapable of reaching high resolutions. Here, we report a ∼2.6-Å structure solved using the antibody-based affinity grid approach with low-concentration Tulane virus purified from a low-yield cell-culture system that has been challenging to standard cryo-EM grid preparation. Quantitative analyses of the structure indicate data and reconstruction quality comparable with the conventional grid preparation method using samples at high concentration.
... For the 200 nm thick section, conventional TEM still produces sharp image with a reasonable contrast (Fig. 5a, left). The current thickness is still within the mean free path of biological section for 200 keV electron, which was measured to be 300 nm (Yan et al., 2015). However, the effect of the focus gradient at the current thickness cannot be avoided (Sun, 2018), affecting the resolution and contrast in TEM. ...
... Our experimental results confirm these effects. For the sample thickness of 200 nm, within the mean free path of 200 keV electron (Yan et al., 2015), the TEM micrograph still yields good contrast with a little of reduced structural details, due to focus gradient. When the sample thickness increases to 400 nm and 600 nm, this reduction becomes significant, for most of the features, terminal. ...
Scanning transmission electron microscopy (STEM) is a powerful imaging technique and has been widely used in current material science research. The attempts of applying STEM (annual dark field (ADF)-STEM or annual bright field (ABF)-STEM) into biological research have been going on for decades while applications have still been limited because of the existing bottlenecks in dose efficiency and non-linearity in contrast. Recently, integrated differential phase contrast (iDPC) STEM technique emerged and achieved a linear phase contrast imaging condition, while resolving signals of light elements next to heavy ones even at low electron dose. This enables successful investigation of beam sensitive materials. Here, we investigate iDPC-STEM advantages in biology, in particular, chemically fixed and resin embedded biological tissues. By comparing results to the conventional TEM, we have found that iDPC-STEM not only shows better contrast but also resolves more structural details at molecular level, including conditions of extremely low dose and minimal heavy-atom staining. We also compare iDPC-STEM with ABF-STEM and found that contrast of iDPC-STEM is even further improved, moderately in lower frequency domains while highly with preserving high frequency biological structural details. For thick sample sections, iDPC-STEM is particularly advantageous. It avoids contrast inversion canceling effects, and by adjusting the depth of focus, fully preserves the contrast of structural details along with the sample. In addition, using depth-sectioning, iDPC-STEM enables resolving in-depth structural variation. Our results suggest that iDPC-STEM have the place and advantages within the future biological research.
... Grids were loaded on to a CM200 (FEG Phillips) electron microscope or a FEI Titan Krios to screen for the presence of virus particles. Ice thickness was estimated for every grid as per the method by Yan et al. [12]. Projection images were obtained for different viruses at 38,000×, 50,000× or 14,000× magnifications at defocus of −3 to −5 µm using a 4K × 4K CCD camera or K2 Summit camera. ...
... We analyzed the morphologies of at least 100 HTNV particles by cryo-EM. We estimated and optimized ice thickness for each virus keeping in mind the diverse range of sizes Yan et al. [12]. About 65% of the particles were round with a mean diameter of 118 nm over a range of 88-148 nm ( Figure 1A). ...
To further understanding of the structure and morphology of the Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus.
... The thickness ( ) of all attenuators was (0.05 cm), and the incident energy of the of molybdenum X-ray was also (17 keV). The transmittance was then written as Lambert's law [28]; ...
This paper studied X-ray attenuation in metals (Al-13, Cu-29, Zr-40). X-ray energy of 17 keV of kα line of molybdenum was directed to metal bars with 0.05 cm thickness. These three metals have differences in their atomic numbers and electronic distributions in the electronic shells; aluminium (Al-13) was chosen as the low atomic number, copper (Cu-29), and zirconium (Zr-40) as the high atomic number. The linear and mass attenuation coefficients, atomic and electronic cross-sections, and electron density for X-ray attenuation through each element were determined experimentally. The results explained a new idea to describe X-ray scattering: the effect of valance and bound electron (electron distribution) of the metals. The metal with more bound electrons in its outermost shell scattered more radiation for a specific range of energy, even though the metal has a less atomic number.
... Sample thickness was estimated by the Beer-Lambert law 45,46 , the value of electron dose per angstrom square was treated as the intensity over vacuum and the elastically scattered electrons counts passing through sample and energy filter as the intensity over sample. The counts of elastic electrons are compensated by 22% due to the presence of coincidence loss 47,48 . ...
Cryo-electron tomography is a major tool used to study the structure of protein complexes in situ. However, the throughput of tilt-series image data collection is still quite low. Here, we show that GisSPA, a GPU accelerated program, can translationally and rotationally localize the target protein complex in cellular lamellae, as prepared with a focused ion beam, using single cryo-electron microscopy images without tilt-series, and reconstruct the protein complex at near-atomic resolution. GisSPA allows high-throughput data collection without the acquisition of tilt-series images and reconstruction of the tomogram, which is essential for high-resolution reconstruction of asymmetric or low-symmetry protein complexes. We demonstrate the power of GisSPA with 3.4-Å and 3.9-Å resolutions of resolving phycobilisome and tetrameric photosystem II complex structures in cellular lamellae, respectively. In this work, we present GisSPA as a practical tool that facilitates high-resolution in situ protein structure determination.
... We took a series of electron micrographs using various defocuses from −1.92 to −0.48 μm and found that the apoferritin particles could show good contrast even at a small defocus of −0.48 μm ( Fig. 2c; Supplementary Fig. 3a), implying that the ice formed was sufficiently thin. We further quantitatively measured the ice thickness based on the inelastic mean free path of electrons 55 and found that the average ice thickness was~33 ± 10 nm, which was thinner than the value of 45 ± 12 nm obtained in the control experiment without HFBI film ( Supplementary Fig. 3b, c). ...
Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
... Determining the thickness of a specimen is nothing new and has been described previously (Cho et al., 2013;Yan et al., 2015;Rice et al., 2018;Suloway et al., 2005). Yet, it has been mostly calculated on high-resolution images and used to monitor sample thickness during an ongoing data collection or to select micrographs based on their sample thickness after ISSN 2059-7983 data acquisition. ...
Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details. This quality-over-quantity approach almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to a lack of high-resolution information. It maximizes the data-collection efficiency and lowers the electron-microscopy time required per data set. This strategy is especially useful if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck.
... Finally, the diffraction data are recorded in a continuous rotation mode as the crystals are continuously rotated under parallel illumination with a high-energy electron beam (typically 200 kV) in a transmission cryo-EM (Hasegawa et al., 2009;Shi et al., 2016). The crystal thickness along the incident beam should be of sub-micrometre dimensions to reduce multiple scattering and absorption (Vulović et al., 2013;Yan et al., 2015;Clabbers & Abrahams, 2018;Latychevskaia & Abrahams, 2019). ...
Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å⁻². The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.
... where λ is the mean free path for electron scattering within the sample [13] and is also named the aperture limited scattering coefficient [12]. We first determined the ice thickness (t) using Λ = 400 nm. Figure 2 shows that the relation in Eq. (2) holds for an estimated ice thickness using Eq. ...
We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope - the first machine of this model, a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer, and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly-coherent 300 kV electron beam to a sample from a cold-field emission gun, and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress, and future plan for running such cryo-EM machines in RIKEN SPring-8 Center.
... To our knowledge, absolute thickness measurements are not routinely determined during single particle experiments, although the objective scattering method has been described (Yan et al., 2015). Determination through tilting is understandably more difficult and unlikely to be performed as a matter of course. ...
Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.
... The change in intensity with tilting a specimen can be used to determine the thickness of the specimen and tilt with respect to the beam. 21 The exit beam intensity is given by a variant of the Beer-Lambert law: ...
Bsoft offers many tools for the processing of tomographic tilt series and the interpretation of tomograms. Since I introduced tomography into Bsoft almost two decades ago, the field has advanced significantly, requiring refinement of old algorithms and development of new ones. The current direct detectors allow us to collect data more efficiently and with better quality, progressing towards automation. The goal is then to also automate alignment of tilt series and reconstruction. I added an estimation of the specimen thickness as well as fiducialless alignment, to augment the existing fiducial‐based alignment. High‐resolution work requires correction for the contrast transfer function, in tomography complicated by the tilted specimen. For this, I developed a method to generate a power spectrum using the whole micrograph, compensating for tilting. This is followed by routine determination of the contrast transfer function, and correction for it during reconstruction. The next steps involve interpretation of the tomogram, either by subtomogram averaging where possible, or by segmentation and modeling otherwise. Such interpretation actually constitutes the main time‐consuming part of tomography and is less amenable to automation compared to the initial reconstruction.
... The thickness of the crystal directly determines the quality of diffraction data (Nannenga and Gonen 2014) due to the mean free path of electron. For 300 kV electron, its mean free path for the vitrified biospecimen is *350 nm, while for 200 kV electron, it is *300 nm (Yan et al. 2015). When the thickness of the crystal is over beyond the mean free path of electron, multiscattering events will become significant and then the diffraction pattern will become difficult to explain. ...
Micro-electron diffraction (MicroED) is an emerging technique to use cryo-electron microscope to study the crystal structures of macromolecule from its micro-/nano-crystals, which are not suitable for conventional X-ray crystallography. However, this technique has been prevented for its wide application by the limited availability of producing good micro-/nano-crystals and the inappropriate transfer of crystals. Here, we developed a complete workflow to prepare suitable crystals efficiently for MicroED experiment. This workflow includes in situ on-grid crystallization, single-side blotting, cryo-focus ion beam (cryo-FIB) fabrication, and cryo-electron diffraction of crystal cryo-lamella. This workflow enables us to apply MicroED to study many small macromolecular crystals with the size of 2–10 μm, which is too large for MicroED but quite small for conventional X-ray crystallography. We have applied this method to solve 2.5 Å crystal structure of lysozyme from its micro-crystal within the size of 10 × 10 × 10 μm³. Our work will greatly expand the availability space of crystals suitable for MicroED and fill up the gap between MicroED and X-ray crystallography.
Electronic supplementary material
The online version of this article (10.1007/s41048-018-0075-x) contains supplementary material, which is available to authorized users.
... This second method has the advantage of not requiring an energy filter. The difference in intensity over vacuum and over sample is described by the Beer-Lambert law, shown in Eq. (2), where d represents the sample thickness, I 0 is intensity over vacuum, I is the intensity over ice, and λ is the mean free path for electron scattering (Yan et al., 2015). ...
... This second method has the advantage of not requiring an energy filter. The difference in intensity over vacuum and over sample is described by the Beer-Lambert law, shown in Eq. (2), where d represents the sample thickness, I 0 is intensity over vacuum, I is the intensity over ice, and λ is the mean free path for electron scattering (Yan et al., 2015). ...
Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.
... For a 300-kV acceleration voltage and 100-nm thickness of a vitrified bio specimen, the dynamic scattering effect can still be neglected because it is smaller than the mean free path (∼350 nm) of 300-keV electrons for the vitrified bio specimen. [64] However, the Ewald sphere effect limits the resolution to 3.8Å according to the formula t · λ /(2 · 0.7), where t is the specimen thickness and λ (= 0.02Å) is the wavelength of 300-keV electrons. [65] The 100-nm focus gradient induces a phase error of π/2 at the resolution of 6.3Å according to the formula ∆χ = πλ ∆Zs 2 , where ∆χ is the phase error and ∆Z is the focus gradient. ...
With 40 years of development, bio-macromolecule cryo-electron microscopy (cryo-EM) has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques: single-particle analysis (SPA), electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air-water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.
... Electrons accelerated at typical TEM voltages can travel 200-300 nm through vitreous ice before a significant amount of inelastic scattering takes place [56,57]. As discussed above, given that cryo-EM contrast is mainly generated by elastically scattered electrons, it is difficult to image much thicker samples with sufficient signal-to-noise ratio even with the help of an energy filter [58,59]. ...
Cryo-electron tomography (cryo-ET) provides high resolution 3D views into cells pristinely preserved by vitrification. Recent technical advances such as direct electron detectors, the Volta phase plate and cryo-focused ion beam milling have dramatically pushed image quality and expanded the range of cryo-ET applications. Cryo-ET not only allows mapping the positions and interactions of macromolecules within their intact cellular context, but can also reveal their in situ structure at increasing resolution. Here, we review how recent work using cutting-edge cryo-ET technologies is starting to provide fresh views into different aspects of cellular biology at an unprecedented level of detail. We anticipate that these developments will soon make cryo-ET a fundamental technique in cell biology. This article is protected by copyright. All rights reserved.
... The reduction in film thickness at the elevated working pressure is most õlikely due to the shorter mean free path for the plasma reactive species, which resulted in less accumulation of precursors. 24,25) By fixing the working pressure at 100 mTorr and applied power at 40 W, the linear growth of HApp films was found at a deposition rate of about 3.99 nm=min [ Fig. 2(b)]. Table I]. ...
The antibacterial performance of the plasma-polymerized (pp) heptylamine thin films loaded with silver nanoparticles was evaluated against the colonization of Escherichia coli and Staphylococcus aureus. The properties including the thickness and chemical composition of the as deposited HApp films were modulated by adjusting plasma parameters. The acquired results showed that the film thickness was controlled in the range of 20 to 400 nm by adjusting deposition time. The subsequent immersion of the HApp thin films in silver nitrate solutions result in the formation of amine-metal complexes, in which the silver nanoparticles were reduced directly on the matrices to form Ag@HApp. The reduction reaction of silver was facilitated by applying NaBH4 as a reducing agent. The results of physicochemical analyses including morphological analysis and ellipsometry revealed that the silver nanoparticles were successfully reduced on the HApp films, and the amount of reduced silver was closely associated which the thickness of the plasma-polymerized films, the concentration of applied metal ions solutions, and the time of immobilization. Regarding the antibacterial performance, the Ag@HApp films reduced by NaBH4 showed antibacterial abilities of 70.1 and 68.2% against E. coli and S. aureus, respectively.
... To minimize multiple scattering in the cryo-STEM, the lamella should be thinner than one mean free path, and thus requires further thinning. For example, the inelastic mean free path of 200-300 keV electrons, typical of the instruments used for cryo-TEM, is a few hundred nanometers in amorphous ice (Vulović et al., 2013;Yan et al., 2015). The goal for the final lamella thickness should then be on the order of 100 nm or less. ...
Scanning transmission electron microscopy (STEM) allows atomic scale characterization of solid-solid interfaces, but has seen limited applications to solid-liquid interfaces due to the volatility of liquids in the microscope vacuum. Although cryo-electron microscopy is routinely used to characterize hydrated samples stabilized by rapid freezing, sample thinning is required to access the internal interfaces of thicker specimens. Here, we adapt cryo-focused ion beam (FIB) "lift-out," a technique recently developed for biological specimens, to prepare intact internal solid-liquid interfaces for high-resolution structural and chemical analysis by cryo-STEM. To guide the milling process we introduce a label-free in situ method of localizing subsurface structures in suitable materials by energy dispersive X-ray spectroscopy (EDX). Monte Carlo simulations are performed to evaluate the depth-probing capability of the technique, and show good qualitative agreement with experiment. We also detail procedures to produce homogeneously thin lamellae, which enable nanoscale structural, elemental, and chemical analysis of intact solid-liquid interfaces by analytical cryo-STEM. This work demonstrates the potential of cryo-FIB lift-out and cryo-STEM for understanding physical and chemical processes at solid-liquid interfaces.
This study employed a comprehensive approach utilizing X-ray diffraction, scanning electron microscopy (computed), computed tomography (CT) three-dimensional scanning, uniaxial compressive testing, acoustic emission (AE), and digital image correlation to investigate the micromorphology, mechanical properties, macro–microscopic fractal characteristics, failure modes, and mineral composition changes in acid–base coal samples from underground reservoirs. The findings from this study indicate that, in acidic environments, calcite undergoes acidolysis to form calcium chloride (CaCl2), whereas kaolinite reacts with alkaline substances to produce albite (Na2Al2Si2O8). The average elastic modulus of the coal samples treated with strong acids or alkalis decreased by 51.08% and 38.17%, respectively, when compared to the naturally dried samples. After strong acid treatment (pH = 2), the post-peak modulus decreased to its lowest value of 1.78 GPa. The fracture threshold in the plastic phase of the acidic coal samples decreased by approximately 60% when compared to that of the naturally dried samples, with the cumulative AE energy reaching a maximum of 2.46 × 1010 aJ. CT image segmentation revealed that with higher H+ concentrations, the sliced fracture fractal dimension increased from 1.01 to 1.25, indicating enhanced fracture disorder. The evolution of the spatial and energy fractal dimensions of the AE, analyzed using the box-counting method and correlation dimension, showed that in strong acidic and alkaline environments, the spatial fractal dimension before the peak failure was higher, pre-disposing the formation of highly disordered and smaller-scale fractures. During the fracture development stage, the time-series energy correlation dimension of the acid–base coal samples experienced two significant fluctuations, with a rapid decrease before the peak, indicating a transition from disorderly expansion to orderly fracturing. In the final stage of fracturing, the strongly acidic samples predominantly developed microcracks. The stronger the acidity or alkalinity, the more likely it is that high-energy (greater than 104 aJ) AE shear events will occur, with the mixed mode of tensile and shear failure tending to concentrate in the areas of high shear strain, thereby enhancing the macroscopic fragmentation orderliness. This research is crucial in regard to the safety assessment of dam bodies or coal pillars in acidic and alkaline coal mines in Northwestern China, particularly for predicting their stability.
Focused ion beam (FIB) is widely used for thinning frozen cells to produce lamellae for cryo-electron microscopy imaging and for protein structures study in vivo. However, FIB damages the lamellae and a quantitative experimental analysis of the damage is lacking. We used a 30-keV gallium FIB to prepare lamellae of a highly concentrated icosahedral virus sample. The viruses were grouped according to their distance from the surface of lamellae and reconstructed. Damage to the approximately 20-nm-thick outermost lamella surface was similar to that from exposure to 16 e-/Å2 in a 300-kV cryo-electron microscope at high-resolution range. The damage was negligible at a depth beyond 50 nm, which was reduced to 30 nm if 8-keV Ga+ was used during polishing. We designed extra steps in the reconstruction refinement to maximize undamaged signals and increase the resolution. The results demonstrated that low-energy beam polishing was essential for high-quality thinner lamellae.
Significance
A systematic investigation of the effects of sample thickness on electron scattering in electron cryo-microscopy (cryo-EM) was previously not feasible. Here, methods are employed to investigate the effects of increasing sample thickness. Near identical protein crystals are used as samples, and microcrystal electron diffraction data are used to assess the effects of thickness. These experiments were conducted using the three most-common accelerating voltages in cryo-EM, and data were compared using the calculated inelastic mean free path. Structures may be determined using crystals up to twice the inelastic mean free path. No coherent information remains at thicknesses over four times the mean free path. This study provides limits for biological specimen thickness with implications for all cryo-EM methods.
Liquid-phase transmission electron microscopy (TEM) has been recently applied to materials chemistry to gain fundamental understanding of various reaction and phase transition dynamics at nanometer resolution. However, quantitative extraction of physical and chemical parameters from the liquid-phase TEM videos remains bottlenecked by the lack of automated analysis methods compatible with the videos’ high noisiness and spatial heterogeneity. Here, we integrate, for the first time, liquid-phase TEM imaging with our customized analysis framework based on a machine learning model called U-Net neural network. This combination is made possible by our workflow to generate simulated TEM images as the training data with well-defined ground truth. We apply this framework to three typical systems of colloidal nanoparticles, concerning their diffusion and interaction, reaction kinetics, and assembly dynamics, all resolved in real-time and real-space by liquid-phase TEM. A diversity of properties for differently shaped anisotropic nanoparticles are mapped, including the anisotropic interaction landscape of nanoprisms, curvature-dependent and staged etching profiles of nanorods, and an unexpected kinetic law of first-order chaining assembly of concave nanocubes. These systems representing properties at the nanoscale are otherwise experimentally inaccessible. Compared to the prevalent image segmentation methods, U-Net shows a superior capability to predict the position and shape boundary of nanoparticles from highly noisy and fluctuating background—a challenge common and sometimes inevitable in liquid-phase TEM videos. We expect our framework to push the potency of liquid-phase TEM to its full quantitative level and to shed insights, in high-throughput and statistically significant fashion, on the nanoscale dynamics of synthetic and biological nanomaterials.
The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30% sucrose cushion, resuspended, and chemically inactivated with 1% glutaraldehyde. A second 30% sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.
At membrane contact sites (MCS) two cellular membranes form tight appositions that play critical roles in fundamental phenomena such as lipid metabolism or Ca2 + homeostasis. The interest for these structures has surged in recent years, bringing about the characterization of a plethora of MCS-resident molecules. How those molecules are structurally organized at MCS remains enigmatic, limiting our understanding of MCS function. Whereas such molecular detail is obscured by conventional electron microscopy sample preparation, cryo-electron tomography (cryo-ET) is uniquely positioned to address this challenge by allowing high resolution imaging of cellular landscapes in close-to-native conditions. Here we briefly review the fundamentals of cryo-ET and how recent developments in this technique are beginning to unveil the molecular architecture of MCS. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.
The plasma membrane and the cell cortex are essential parts of the eukaryotic cell. The plasma membrane delimitates the cell and mediates communication with the outside. The cell cortex is the submembrane cytoskeleton shaping the cell and is able to reorganize for the passage of material. To study events at and near the plasma membrane, cryoelectron microscopy (cryo-EM) may be used. Most intact cells are too thick for direct cryo-EM imaging. Generating cell-free membrane patches could be a means to study features at the plasma membrane. Here we present an unroofing method, termed iMEM (isolation of membrane patches for cryo-EM) where the plasma membrane is isolated directly on an EM grid. The in situ isolation of membrane patches has several advantages: it is a one-step procedure providing a higher throughput than focused-ion beam cryomilling. It enables the time-precise control over biochemical events before cryofixation.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune
defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly
understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted
in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the
first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments
could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four
human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of
antibody therapeutics with enhanced efficacy.
Background
Cryo-electron microscopy is an excellent method for the structural analysis of biological materials. Advantage of its use over conventional electron microscopy techniques is the preservation of the sample in a near-native, hydrated state. To achieve the analysis with greatly improved structural details, optimization of various parameters involved in sample vitrification is required. Most considerable parameter is the thickness of ice: thick and thin layers are ideally in favor for larger and smaller target objects.
Findings
We measured the thickness of vitreous ice from different types of widely used holey carbon grids using cryo-EM and electron energy loss spectroscopy. It showed that Quantifoil grids are suitable for the structural analysis of large biological macromolecules (>100 nm in size), whereas the use of lacey and C-flat grids are ideal for smaller particles.
Conclusions
This report provides informative details that may help increasing chances of obtaining optimal vitreous ice for various biological objects with different sizes, hence facilitate the successful application of cryo-electron microscopy.
In the Python world, NumPy arrays are the standard representation for numerical data and enable efficient implementation of numerical computations in a high-level language. As this effort shows, NumPy performance can be improved through three techniques: vectorizing calculations, avoiding copying data in memory, and minimizing operation counts.
Small-scale testing is extensively used to study the effects of size on plasticity or characterise plastic deformation of brittle materials, where cracking is suppressed on the microscale. Geometrical and experimental constraints have been shown to affect small-scale defor- mation and efforts are underway to understand these better. However, current analytical techniques tend to possess high resolution in only one or two dimensions, impeding a detailed analysis of the entire deformed volume. Here electron backscattered diffraction in three dimensions is presented as a way of characterising three-dimensional (3-D) deformation at high spatial resolution. It is shown that, by reconstruction of compressed and then successively sliced and indexed MgO micropillars, this 3-D technique yields information comple- mentary to l-Laue diffraction or electron microscopy, allowing a correlation of experimental artefacts and the distribution of plasticity. In addition, deformation features which are difficult to visualise by standard scanning electron microscopy are easily detected, for exam- ple where only small surface traces are produced or minimal plastic strain can be introduced before failure in brittle materials.
High-resolution electron tomography from a tilt series of transmission electron microscopy images requires an accurate image alignment procedure in order to maximise the resolution of the tomogram. This is the case in particular for ultra-high resolution where even very small misalignments between individual images can dramatically reduce the fidelity of the resultant reconstruction. A tomographic-reconstruction based and marker-free method is proposed, which uses an iterative optimisation of the tomogram resolution. The method utilises a search algorithm that maximises the contrast in tomogram sub-volumes. Unlike conventional cross-correlation analysis it provides the required correlation over a large tilt angle separation and guarantees a consistent alignment of images for the full range of object tilt angles. An assessment based on experimental reconstructions shows that the marker-free procedure is competitive to the reference of marker-based procedures at lower resolution and yields sub-pixel accuracy even for simulated high-resolution data.
The theoretical and experimental bases for quantitative electron microscopy of frozen-hydrated specimens are described, with special considerations of energy filtration to improve the images. The elastic and inelastic scattering from molecules in vacuum and in ice are calculated, and simple methods to approximate scattering are introduced. Multiple scattering calculations are used to describe the scattering from vitreous ice and to predict the characteristics of images of frozen-hydrated molecules as a function of ice thickness and accelerating voltage. Energy filtration is predicted to improve image contrast and signal-to-noise ratio. Experimental values for the inelastic scattering of ice, the energy spectrum of thick ice, and the contrast of biological specimens are determined. The principles of compensation for the contrast transfer function are presented. Tobacco mosaic virus is used to quantify the accuracy of interpreting image intensities to derive the absolute mass, mass per unit length, and internal mass densities of biological molecules. It is shown that compensation for the contrast transfer function is necessary and sufficient to convert the images into accurate representations of molecular density. At a resolution of 2 nm, the radial density reconstructions of tobacco mosaic virus are in quantitative agreement with the atomic model derived from X-ray results.
Cryo-electron microscopy of vitrified specimens was just emerging as a practical method when Richard Henderson proposed that we should teach an EMBO course on the new technique. The request seemed to come too early because at that moment the method looked more like a laboratory game than a useful tool. However, during the months which ellapsed before the start of the course, several of the major difficulties associated with electron microscopy of vitrified specimens found surprisingly elegant solutions or simply became non-existent. The course could therefore take place under favourable circumstances in the summer of 1983. It was repeated the following years and cryo-electron microscopy spread rapidly. Since that time, water, which was once the arch enemy of all electronmicroscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.
We have shown that a scanning transmission electron microscope with a high brightness field emission source is capable of obtaining better than 3 A resolution using 30 to 40 keV electrons. Elastic dark field images of single atoms of uranium and mercury are shown which demonstrate this fact as determined by a modified Rayleigh criterion. Point-to-point micrograph resolution between 2.5 and 3.0 A is found in dark field images of micro-crystallites of uranium and thorium compounds. Furthermore, adequate contrast is available to observe single atoms as light as silver.
1. Introduction 308
2. Electron microscopy 311
2.1 Specimen preparation 311
2.2 The electron microscope 311
2.3 Acceleration voltage, defocus, and the electron gun 312
2.4 Magnification and data collection 313
3. Digitisation and CTF correction 317
3.1 The patchwork densitometer 318
3.2 Particle selection 320
3.3 Position dependent CTF correction 321
3.4 Precision of CTF determination 321
4. Single particles and angular reconstitution 323
4.1 Preliminary filtering and centring of data 323
4.2 Alignments using correlation functions 324
4.3 Choice of first reference images 324
4.4 Multi-reference alignment of data 325
4.5 MSA eigenvector/eigenvalue data compression 328
4.6 MSA classification 330
4.7 Euler angle determination (‘angular reconstitution’) 332
4.8 Sinograms and sinogram correlation functions 332
4.9 Exploiting symmetry 335
4.10 Three-dimensional reconstruction 337
4.11 Euler angles using anchor sets 339
4.12 Iterative refinements 339
5. Computational hardware/software aspects 341
5.1 The ( IMAGIC ) image processing workstation 342
5.2 Operating systems and GUIs 342
5.3 Computational logistics 344
5.4 Shared memory machines 344
5.5 Farming on loosely coupled computers 346
5.6 Implementation using MPI protocol 347
5.7 Software is what it's all about 347
6. Interpretation of results 348
6.1 Assessing resolution: the Fourier Shell Correlation 348
6.2 Influence of filtering 351
6.3 Rendering 351
6.4 Searching for known sub-structures 352
6.5 Interpretation 353
7. Examples 353
7.1 Icosahedral symmetry: TBSV at 5·9 Å resolution 354
7.2 The D6 symmetrical worm hemoglobin at 13 Å resolution 356
7.3 Functional states of the 70S E. coli ribosome 357
7.4 The 50S E. coli ribosomal subunit at 7·5 Å resolution 359
8. Perspectives 361
9. Acknowledgements 364
10. References 364
In the past few years, electron microscopy (EM) has established itself as an important – still upcoming – technique for studying the structures of large biological macromolecules. EM is a very direct method of structure determination that complements the well-established techniques of X-ray crystallography and NMR spectroscopy. Electron micrographs record images of the object and not just their diffraction patterns and thus the classical ‘phase’ problem of X-ray crystallography does not exist in EM. Modern microscopes may reach resolution levels better than ∼ 1·5 Å, which is more than sufficient to elucidate the polypeptide backbone in proteins directly. X-ray structures at such resolution levels are considered ‘excellent’. The fundamental problem in biological EM is not so much the instrumental resolution of the microscopes, but rather the radiation sensitivity of the biological material one wants to investigate. Information about the specimen is collected in the photographic emulsion with the arrival of individual electrons that have (elastically) interacted with the specimen. However, many electrons will damage the specimen by non-elastic interactions. By the time enough electrons have passed through the object to produce a single good signal-to-noise (SNR) image, the biological sample will have been reduced to ashes. In contrast, stable inorganic specimens in material science often show interpretable details down to the highest possible instrumental resolution.
The inelastic mean free path of 120 keV electrons in vitrified ice layers has been determined in an energy-filtering TEM. From the ratio of the unfiltered and zero-loss-filtered image intensities recorded with a slow-scan CCD camera, the relative sample thickness t/Lambda can be calculated. For calibration, the geometric ice thickness was measured by imaging a tilted view of a cylindrical hole which had been burnt into the ice layer. The total inelastic mean free path was found to be 161 nm, and the partial inelastic mean free path for an acceptance angle of 4.2 mrad was 232 nm. These results were built into a standard protocol for use in cryo-electron microscopy allowing on-line measurements of local ice-layer thicknesses by zero-loss-filtered/unfiltered imaging.
Python is an interpreted language with expressive syntax, which transforms itself into a high-level language suited for scientific and engineering code. Some of its features include a liberal open source license, ability to run on many platforms, powerful interactive interpreter, ability to expand with earlier compiled code, ability to interact with a wide variety of other software, and a large number of library modules. An important factor in the utility of Python as a computing language is its clear syntax, which can make code easy to understand and maintain. This language also contributes to the construction of maintainable code by separating code into logical groups such as modules, class, and functions, in addition to offering clean syntax. Python can be easily extended with a large C-API for calling Python functionality from C programming language, connecting to non-Python compiled code, and extending the language itself by creating new Python types in C language.
Electron tomography is a method whereby a three-dimensional reconstruction of a nanoscale object is obtained from a series of projected images measured in a transmission electron microscope. We developed an electron-diffraction method to measure the tilt and azimuth angles, with Kikuchi lines used to align a series of diffraction patterns obtained with each image of the tilt series. Since it is based on electron diffraction, the method is not affected by sample drift and is not sensitive to sample thickness, whereas tilt angle measurement and alignment using fiducial-marker methods are affected by both sample drift and thickness. The accuracy of the diffraction method benefits reconstructions with a large number of voxels, where both high spatial resolution and a large field of view are desired. The diffraction method allows both the tilt and azimuth angle to be measured, while fiducial marker methods typically treat the tilt and azimuth angle as an unknown parameter. The diffraction method can be also used to estimate the accuracy of the fiducial marker method, and the sample-stage accuracy. A nano-dot fiducial marker measurement differs from a diffraction measurement by no more than ±1°.
Cryo-Electron Tomography (CET) is the only available technique capable of characterizing the structure of biological macromolecules in conditions close to the native state. With the advent of subtomogram averaging, as a post-processing step to CET, resolutions in the (sub-)nanometer range have become within reach. In addition to advances in instrumentation and experiments, the reconstruction scheme has improved by inclusion of more accurate contrast transfer function (CTF) correction methods, better defocus estimation, and better alignments of the tilt-series and subtomograms. To quantify the importance of each contribution, we have split the full process from data collection to reconstruction into different steps. For the purpose of evaluation we have acquired tilt-series of ribosomes in such a way that we could precisely determine the defocus of each macromolecule. Then, we simulated tilt-series using the InSilicoTEM package and applied tomogram reconstruction and subtomogram averaging. Through large scale simulations under different conditions and parameter settings we find that tilt-series alignment is the resolution limiting factor for our experimental data. Using simulations, we find that when this alignment inaccuracy is alleviated, tilted CTF correction improves the final resolution, or equivalently, the same resolution can be achieved using less particles. Furthermore, we predict from which resolution onwards better CTF correction and defocus estimation methods are required. We obtain a final average using 3198 ribosomes with a resolution of 2.2nm on the experimental data. Our simulations suggest that with the same number of particles a resolution of 1.2nm could be achieved by improving the tilt-series alignment.
Choosing the correct error injection technique is of primary importance in simulation-based design and evaluation of robust systems that are resilient to soft errors. Many low-level (e.g., flip-flop-level) error injection techniques are generally used for small systems due to long execution times and significant memory requirements. High-level error injections at the architecture or memory levels are generally fast but can be inaccurate. Unfortunately, there exists very little research literature on quantitative analysis of the inaccuracies associated with high-level error injection techniques. In this paper, we use simulation and emulation results to understand the accuracy tradeoffs associated with a variety of high-level error injection techniques. A detailed analysis of error propagation explains the causes of high degrees of inaccuracies associated with error injection techniques at higher levels of abstraction.
Three-dimensional reconstructions of microstructures produced by Focused Ion Beam (FIB) milling usually assume a uniform slice thickness with flat and parallel surfaces. Measurement of the actual slice thickness and profile is difficult, and is often simply ignored. This paper reports the use of artificial 3D structures of known geometry to enable the full 3D profile of a sequence of slices produced by FIB to be measured for the first time. A transient period at the beginning of a milling process is observed in which the actual slice thickness varies by as much as +/−50% from the target thickness (with significantly greater error near the base of the slice), before settling to a +/−20% variation as the milling progresses. Although SEM images appear to show flat milled surfaces perpendicular to the top surface, the development of a curved, tapering milled surface is also observed. This profile is then maintained through the milling process with the bottom of the slice lagging the top by up to three slice thicknesses.
Bright Field (BF) electron tomography (ET) has been widely used in the
life sciences to characterize biological specimens in 3D. While BF-ET is
the dominant modality in the life sciences it has been generally avoided
in the physical sciences due to anomalous measurements in the data due
to a phenomenon called "Bragg scatter" - visible when crystalline
samples are imaged. These measurements cause undesirable artifacts in
the reconstruction when the typical algorithms such as Filtered Back
Projection (FBP) and Simultaneous Iterative Reconstruction Technique
(SIRT) are applied to the data. Model based iterative reconstruction
(MBIR) provides a powerful framework for tomographic reconstruction that
incorporates a model for data acquisition, noise in the measurement and
a model for the object to obtain reconstructions that are qualitatively
superior and quantitatively accurate. In this paper we present a novel
MBIR algorithm for BF-ET which accounts for the presence of anomalous
measurements from Bragg scatter in the data during the iterative
reconstruction. Our method accounts for the anomalies by formulating the
reconstruction as minimizing a cost function which rejects measurements
that deviate significantly from the typical Beer's law model widely
assumed for BF-ET. Results on simulated as well as real data show that
our method can dramatically improve the reconstructions compared to FBP
and MBIR without anomaly rejection, suppressing the artifacts due to the
Bragg anomalies.
Serial block-face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack-acquisition time to several days. Here, we describe a method for controlling the ion-beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted-beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion-mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in-column detector is explored.
Accurate modeling of image formation in cryo-electron microscopy is an important requirement for quantitative image interpretation and optimization of the data acquisition strategy. Here we present a forward model that accounts for the specimen's scattering properties, microscope optics, and detector response. The specimen interaction potential is calculated with the isolated atom superposition approximation (IASA) and extended with the influences of solvent's dielectric and ionic properties as well as the molecular electrostatic distribution. We account an effective charge redistribution via the Poisson-Boltzmann approach and find that the IASA-based potential forms the dominant part of the interaction potential, as the contribution of the redistribution is less than 10 %. The electron wave is propagated through the specimen by a multislice approach and the influence of the optics is included via the contrast transfer function. We incorporate the detective quantum efficiency of the camera due to the difference between signal and noise transfer characteristics, instead of using only the modulation transfer function. The full model was validated against experimental images of 20S proteasome, hemoglobin, and GroEL. The simulations adequately predict the effects of phase contrast, changes due to the integrated electron flux, thickness, inelastic scattering, detective quantum efficiency and acceleration voltage. We suggest that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected. All simulation parameters are based on physical principles and, when necessary, experimentally determined.
Scattering contrast measurements were performed on thin films of amorphous carbon and polycrystalline Au, as well as single-crystal MgO nanocubes. Based on the exponential absorption law, mass-thickness can be obtained within 10% accuracy by measuring the incident and transmitted intensities in the same image. For mass-thickness measurement of a thin amorphous specimen, a small collection semiangle improves the measurement sensitivity, whereas for the measurement of polycrystalline or single-crystal specimens, a large collection semiangle should be used to reduce diffraction-contrast effects. EELS thickness measurements on MgO nanocubes suggest that the Kramers-Kronig sum-rule method (with correction for plural and surface scattering) gives 10% accuracy at medium collection semiangles but overestimates the thickness at small collection semiangles, due to underestimation of the surface-mode scattering. The log-ratio method, with a formula for inelastic mean free path proposed by Malis et al. (1988), provides 10% accuracy at small collection semiangle, while that proposed by Iakoubovskii et al. (2008a) is preferable for medium and large collection semiangles. As a result of this work, we provide recommendations of preferred methods and conditions for local-thickness measurement in the TEM.
We discuss measurement of the local thickness t of a transmission microscope specimen from the log-ratio formula t = lambda ln (It/I0) where It and I0 are the total and zero-loss areas under the electron-energy loss spectrum. We have measured the total inelastic mean free path lambda in 11 materials of varying atomic number Z and have parameterized the results in the form lambda = 106F (E0/Em)/ln (2 beta E0/Em) where F = (1 + E0/1,022)/(1 + E0/511)2, the incident energy E0 is in keV, the spectrum collection semiangle beta is in mrad, and Em = 7.6Z0.36. This formulation should allow absolute thickness to be determined to an accuracy of +/- 20% in most inorganic specimens.
Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions. The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy. Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method.
The use of fully automated data collection methods for electron tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self-consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post-cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by pre-irradiation. We believe that the accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Furthermore neither the choice of embedding resin nor microwave post-curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal pre-irradiation coupled with low-temperature automated data collection greatly reduces shrinkage and should result in high-quality data for three-dimensional reconstructions.
We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
Using electron microscopy, the thickness of ice-embedded vesicles is estimated examining tilted and untilted views and assuming an ellipsoidal shape of the vesicles that appear to be circular in the untilted view. Another thickness measure is obtained from the ratio of the unfiltered and zero-loss-filtered image intensities of the vesicle. From these two measurements, the mean free path A for inelastic scattering of electrons in ice is calculated as 203 +/- 33 nm for 120 kV acceleration voltage. It is found that vesicles in thin ice films (< or = 1.5 lambda) significantly protrude out of the ice film. Due to surface tension the shape becomes an oblate ellipsoid. In holes covered with a thick ice film (> or = 3 lambda) and strong thickness gradients, vesicles are predominantly found in regions where the ice thickness is appropriate for their size. Also, a way of imaging the most probable loss under low-dose conditions involving thickness measurement is proposed. Even at large ice thicknesses zero-loss filtering always gives better image contrast. Most probable loss imaging can only help where there is no intensity in the zero-loss image, at very large thicknesses (lambda > 8).
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.
Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.
An image alignment method for electron tomography is presented which is based on cross-correlation techniques and which includes a simultaneous refinement of the tilt geometry. A coarsely aligned tilt series is iteratively refined with a procedure consisting of two steps for each cycle: area matching and subsequent geometry correction. The first step, area matching, brings into register equivalent specimen regions in all images of the tilt series. It determines four parameters of a linear two-dimensional transformation, not just translation and rotation as is done during the preceding coarse alignment with conventional methods. The refinement procedure also differs from earlier methods in that the alignment references are now computed from already aligned images by reprojection of a backprojected volume. The second step, geometry correction, refines the initially inaccurate estimates of the geometrical parameters, including the direction of the tilt axis, a tilt angle offset, and the inclination of the specimen with respect to the support film or specimen holder. The correction values serve as an indicator for the progress of the refinement. For each new iteration, the correction values are used to compute an updated set of geometry parameters by a least squares fit. Model calculations show that it is essential to refine the geometrical parameters as well as the accurate alignment of the images to obtain a faithful map of the original structure.
A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film into the microscope dramatically increases the contamination rate, particularly in the first 3 h after the insertion of the film box. In the absence of film, we were able to reproducibly demonstrate that the contamination rate can be limited to a rate of approximately 1 angstrom/h providing reassurance that contamination will not be a major limiting factor for long term cryoEM experiments if a CCD camera is used for the imaging.
EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.
We present a fully automated method for three-dimensional (3D) elemental analysis demonstrated using a ceramic sample of chemistry (Ca)MgTiO(x). The specimen is serially sectioned by a focused ion beam (FIB) microscope, and energy-dispersive X-ray spectrometry (EDXS) is used for elemental analysis of each cross-section created. A 3D elemental model is reconstructed from the stack of two-dimensional (2D) data. This work concentrates on issues arising from process automation, the large sample volume of approximately 17 x 17 x 10 microm(3), and the insulating nature of the specimen. A new routine for post-acquisition data correction of different drift effects is demonstrated. Furthermore, it is shown that EDXS data may be erroneous for specimens containing voids, and that back-scattered electron images have to be used to correct for these errors.
Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.
Matplotlib is a 2D graphics package used for Python for application development, interactive scripting, and publication-quality image generation across user interfaces and operating systems. The latest release of matplotlib runs on all major operating systems, with binaries for Macintosh's OS X, Microsoft Windows, and the major Linux distributions. Matplotlib has a Matlab emulation environment called PyLab, which is a simple wrapper of the matplotlib API. Matplotlib provides access to basic GUI events such as button_press_event, mouse_motion_event and can also be registered with those events to receive callbacks. Event handling code written in matplotlib works across many different GUIs. It supports toolkits for domain specific plotting functionality that is either too big or too narrow in purpose for the main distribution. Matplotlib has three basic API classes, including, FigureCanvasBase, RendererBase and Artist.
Accurate measurement of relative tilt and azimuth angles in electron tomography: a comparison of fiducial marker method with electron diffraction. The Review of scientific instruments
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An improved holey carbon film for cryo-electron microscopy. Microscopy and microanalysis : the official journal of Microscopy Society of America
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Author manuscript; available in PMC
J Struct Biol. Author manuscript; available in PMC 2016 November 01.