Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Accurate determination of local defocus and specimen tilt in electron microscopy

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.     

A contamination reducing method by ion beam bombardment of the specimen in high resolution electron microscopy

The theory of contamination and a contamination reducing method are discussed on the basis of time dependent micrograph series and their tilted images for determining contamination.For a high current density of an electron probe in the field emission scanning electron microscope, it is observed that contaminated cones are formed in proportion to the exposure time of an electron beam. From the measurement of the contamination layer thickness and its area, the contamination rate and time dependent shape are formulated, mainly depending on the cross-section and current density together with the average lifetime of adsorption molecules.It is found that the contamination rate and stray contamination of outgassing molecules forming part of the specimen are effectively reduced by a pre-bombardment of argon ions on the surfaces of specimens. The contamination rate is reduced to a small extent (5%) using the present method.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Improved specimen reconstruction by Hilbert phase contrast tomography

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

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.     

The ups and downs of beam damage,contrast and noise in biological electron microscopy

A personal account of the early problems associated with contrast in images obtained by electron microscopy from biological specimens is presented, together with the effects of electron beam damage. The author's experiences with different types of electron microscope as well as problems of contrast enhancement is described. A short account is given of the physical effects occuring during specimen preparation and their relation to structural preservation when attempting to achieve atomic resolution. Recent developments in biological electron microscopy are also discussed with a view to future trends.     

Variations of the three-dimensional structure of the Escherichia coli ribosome in the range of overlap views. An application of the methods of multicone and local single-cone three-dimensional reconstruction.

Electron microscopic techniques are among the most important tools for obtaining structural information of biological specimens. However, the three-dimensional (3D) structural analysis of asymmetrical specimens that do not form crystalline sheets has traditionally presented serious methodological obstacles to its accomplishment. One of the fundamental questions to be addressed in this type of structural study is in what way, and to what degree, does the 3D structural conformation depend on the orientation of the specimen with respect to the electron microscopic support films. As a step in studying this problem, we have analyzed the variations of the 3D structure of the Escherichia coli 70S monosome by performing four different 3D reconstructions of the 70S monosome from subsets of images in the so-called overlap range of views. These subsets were selected according to a multivariate statistical analysis performed on the total population of overlap-range specimen images. A certain amount of structural variability exists among the 3D reconstructions, although many of the main morphological characteristics, as the relative orientation between the ribosomal subunits, remain unchanged. We have also generalized the random conical reconstruction technique (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. J. Microsc. 146: 113-136) to include those cases where the specimen exhibits a rocking behavior with respect to the support. The resulting Multicone Reconstruction Technique has been applied to computer-generated images as well as the E. coli 70S monosome images from part of the overlap range of views.     

Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.     

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.     

Calculation of tilt angles for crystal specimen orientation adjustment using double-tilt and tilt-rotate holders

In this communication we wish to present a group of new equations which can be used to calculate the tilt angle for crystal specimen orientation adjustment in the transmission electron microscope. The experiments were concerned with double-tilt and tilt-rotate holders and the new equations deduced using matrix geometry. The specimen orientation adjustment using the tilt angles calculated by these equations is considered to be more convenient and less time-consuming than following the Kikuchi map method. Our method avoids the difficulties associated with orientation adjustment of severely strained and small grain size specimens using the Kikuchi map procedure. The algorithms for deducing the new equations, together with an experimental example using the equations, are described.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

A system for the three-dimensional construction,manipulation and display of microbiological models

A system is described for building up serial sections into a three dimensional structure, incorporating density, that can be displayed and then further manipulated by rotation about three orthogonal axes. The initial application was to produce a computer model of a protein structure and to compare the diverse images obtained from rotation with the two dimensional images observed in related electron micrographs. To obtain sufficient contrast in the electron microscope images of protein structures, the specimens need to be stained and since this can cause some deformation of the observed images, it is also necessary to simulate the possible effects of stain on the protein model. Because of the need to compare numerous orientations of the combined model, techniques are available either for speeding up the comparison or for obtaining better accuracy. The methods have been applied to the interpretation of electron micrograph images of microbiological specimens, where the three dimensional structure of the specimen is an important aid in understanding its biological function, but the techniques are also applicable to more general serial reconstruction requirements.     

High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein

We describe the electron microscopy of a crystalline assembly of an alpha-helical coiled-coil protein extracted from the ootheca of the praying mantis. Electron diffraction patterns of unstained crystals show crystal lattice sampling of the coiled-coil molecular transform to a resolution beyond 1.5 A. Using a "spot-scan" method of electron imaging, micrographs of unstained crystals have been obtained that visibly diffract laser light from crystal spacings as small as 4.3 A. A projection map was calculated to 4 A using electron diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1 A alpha-helical repeat, the first time this type of modulation has been revealed by electron microscopy. The crystals have p2 plane group symmetry with a = 92.4 A, b = 150.7 A, y = 92.4 degrees. Examination of tilted specimens shows that c is approximately 18 A, indicating that the unit cell is only one molecule thick. A preliminary interpretation shows tightly packed molecules some 400 A long lying with their long axes in the plane of the projection. The molecules have a coiled-coil configuration for most of their length. The possible modes of packing of the molecules in three dimensions are discussed.     

Negative staining of freshwater bacterioneuston sampled directly with electron microscope specimen support grids

A technique for observation of surface microlayer bacteria (bacterioneuston) is described, utilizing direct sampling of the air-water interface with carbon-stabilized electron microscope specimen support grids, followed by negative staining and transmission electron microscopy. The method resulted in excellent preservation of forms of microcolonial association, regular surface arrays, surface appendages, and prosthecae in the bacterioneuston of a freshwater pond.     

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens.

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens was studied by non-destructive uniaxial compression to 0.4% strain using cylindrical specimens with different sizes and length-to-diameter ratios, and by comparing cubic and cylindrical specimens with the same cross-sectional area. Both the length and the cross-sectional area of the specimen had a highly significant influence on the mechanical behaviour (p less than 0.0001). Within the actual range of length (2.75-11.0 mm) the normalized stiffness (Young's modulus) was related nearly linearly to the specimen length. This dependency on specimen length is suggested to be caused mainly by structural disintegrity of the trabecular specimens near the surface. The normalized stiffness (Young's modulus) was also positively correlated to the cross-sectional area. This dependency on cross-sectional area is probably due to friction-induced stress inhomogeneity at the platen-specimen interface. A cube with side length 6.5 mm or a cylindrical specimen with 7.5 mm diameter and 6.5 mm length are suggested as standard specimens for comparative studies on trabecular bone mechanics.     

A deep learning-based approach for detecting plant organs from digitized herbarium specimen images

Herbarium specimens are excellent sources of botanical information to facilitate understanding and monitoring the evolution of plants and their effects on global climate change. Globally, many herbaria have undertaken digitization projects of herbarium specimens to preserve them and make them accessible in online repositories to botanists and ecologists. Automated detection of plant organs such as plant leaves, buds, flowers, and fruits on the digitized herbarium specimen images provides valuable information in various scientific contexts. We developed a deep learning approach based on the refined YOLO-V3 approach to detect plant organs within the digitized herbarium specimen images effectively. The proposed approach combines ResNet and DenseNet architectures to improve feature extraction capabilities. Also, a new scale of feature map is added to the existing scales to address the problem of YOLO-V3's low performance in detecting small plant organs. The experimental results demonstrate that our proposed approach can detect organs of different sizes within different specimens, where the precision and recall reached 94.2% and 95.5%, respectively.     

Three-dimensional structure of T3 connector purified from overexpressing bacteria.

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.