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Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

Manganese-based Prussian Blue, Na2-δ Mn[Fe(CN)6 ] (MnPB), is a good candidate for sodium-ion battery cathode materials due to its high capacity. However, it suffers from severe capacity decay during battery cycling due to the destabilizing Jahn-Teller distortions it undergoes as Mn2+ is oxidized to Mn3+ . Herein, the structure is stabilized by a thin epitaxial surface layer of nickel-based Prussian Blue (Na2-δ Ni[Fe(CN)6 ]). The one-pot synthesis relies on a chelating agent with an unequal affinity for Mn2+ and Ni2+ ions, which prevents Ni2+ from reacting until the Mn2+ is consumed. This is a new and simpler synthesis of core-shell materials, which usually needs several steps. The material has an electrochemical capacity of 93 mA h g-1 , of which it retains 96 % after 500 charge-discharge cycles (vs. 37 % for MnPB). Its rate capability is also remarkable: at 4 A g-1 (ca. 55 C) it can reversibly store 70 mA h g-1 , which is also reflected in its diffusion coefficient of ca. 10-8 cm2 s-1 . The epitaxial outer layer appears to exert an anisotropic strain on the inner layer, preventing the Jahn-Teller distortions it normally undergoes during de-sodiation.

In traditional non-flammable electrolytes a trade-off always exists between non-flammability and battery performance. Previous research focused on reducing free solvents and forming anion-derived solid-electrolyte interphase. However, the contribution of solvated anions in boosting the stability of electrolyte has been overlooked. Here, we resolve this via introducing anions into Li

The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redβ, a defining member of the Redβ/RecT protein family.

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria., Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled transcription complexes. Here the authors provide mechanistic insights into this process by determining the cryo-EM structures of the Bacillus subtilis RNA polymerase (RNAP) elongation complex and the RNAP-HelD transcription recycling complex and propose a model of HelD catalysed transcription recycling.

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes distressed RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, β and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate ‘half-up’ state to a condensed ‘down’ state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory ‘up’ state.

Lithium metal has been considered the "holy grail" anode material for rechargeable batteries despite the fact that its dendritic growth and low Coulombic efficiency (CE) have crippled its practical use for decades. Its high chemical reactivity and low stability make it difficult to explore the intrinsic chemical and physical properties of the electrochemically deposited lithium (EDLi) and its accompanying solid electrolyte interphase (SEI). To prevent the dendritic growth and enhance the electrochemical reversibility, it is crucial to understand the nano- and mesostructures of EDLi. However, Li metal is very sensitive to beam damage and has low contrast for commonly used characterization techniques such as electron microscopy. Inspired by biological imaging techniques, this work demonstrates the power of cryogenic (cryo)-electron microscopy to reveal the detailed structure of EDLi and the SEI composition at the nanoscale while minimizing beam damage during imaging. Surprisingly, the results show that the nucleation-dominated EDLi (5 min at 0.5 mA cm-2) is amorphous, while there is some crystalline LiF present in the SEI. The EDLi grown from various electrolytes with different additives exhibits distinctive surface properties. Consequently, these results highlight the importance of the SEI and its relationship with the CE. Our findings not only illustrate the capabilities of cryogenic microscopy for beam (thermal)-sensitive materials but also yield crucial structural information on the EDLi evolution with and without electrolyte additives.

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

We report on progress of employing the Kepler workflow engine to prototype “end-to-end” application integration workflows that concern data coming from microscopes deployed at the National Center for Microscopy Imaging Research (NCMIR). This system is built upon the mature code base of the Cell Centered Database (CCDB) and integrated rule-oriented data system (IRODS) for distributed storage. It provides integration with external projects such as the Whole Brain Catalog (WBC) and Neuroscience Information Framework (NIF), which benefit from NCMIR data. We also report on specific workflows which spawn from main workflows and perform data fusion and orchestration of Web services specific for the NIF project. This “Brain data flow” presents a user with categorized information about sources that have information on various brain regions.

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide-field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast-scanning, multiphoton, confocal microscope fitted with a motorized, high-precision stage and custom-developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.

Alignment of the individual images of a tilt series is a critical step in obtaining high-quality electron microscope reconstructions. We report on general methods for producing good alignments, and utilizing the alignment data in subsequent reconstruction steps. Our alignment techniques utilize bundle adjustment. Bundle adjustment is the simultaneous calculation of the position of distinguished markers in the object space and the transforms of these markers to their positions in the observed images, along the bundle of particle trajectories along which the object is projected to each EM image. Bundle adjustment techniques are general enough to encompass the computation of linear, projective or nonlinear transforms for backprojection, and can compensate for curvilinear trajectories through the object, sample warping, and optical aberration. We will also report on new reconstruction codes and describe our results using these codes.

We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z -direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 μm thick.

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD’s) to increase the signal to noise as compared with CCD’s. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

High quality tomographic reconstruction from electron microscope images requires precise alignment of the images. This cannot be accomplished by simple 2D image transformations because the electron trajectories through the object are curvilinear. Accordingly, the alignment process requires an inferred object position and, in addition, nonlinear projection maps for each image. This may be accomplished by means of a generalized bundle adjustment when a consistent set of point-like objects is visible in each EM image. Alternatively, alignment may be accomplished by an extension of the method of occluded contours when a consistent set of linear features, such as projected outlines of surfaces can be identified. In the first case, we report on new results derived from multiple tilt series data, and in the second we report on high quality alignment and reconstruction when the number of point features is insufficient for proper alignment.

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120-400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721-739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 microm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.

In a longstanding effort to overcome limits of film and the charge coupled device (CCD) systems in electron microscopy, we have developed a radiation-tolerant system that can withstand direct electron bombardment. A prototype Direct Detection Device (DDD) detector based on an Active Pixel Sensor (APS) has delivered unprecedented performance with an excellent signal-to-noise ratio (approximately 5/1 for a single incident electron in the range of 200-400 keV) and a very high spatial resolution. This intermediate size prototype features a 512x550 pixel format of 5μm pitch. The detector response to uniform beam illumination and to single electron hits is reported. Radiation tolerance with high-energy electron exposure is also impressive, especially with cooling to -15 °C. Stable performance has been demonstrated, even after a total dose of 3.3x10 6 electrons/pixel. The characteristics of this new detector have exciting implications for transmission electron microscopy, especially for cryo-EM as applied to biological macromolecules.

Publisher Summary Charge-coupled device (CCD) invented in 1970, soon became the sensor of choice in many imaging applications, particularly for video cameras and camcorders. This chapter reviews current efforts to scale up lens-coupled CCD camera and make a system capable of exceeding the spatial resolution of film, while maintaining single-electron sensitivity. This lens-coupled CCD system represents the current state-of-the-art in CCD-based systems, and it also demonstrates the great engineering effort required to achieve these key performance benchmarks when the detector is based on a resolution-limiting scintillation screen. The chapter discusses the development of a parallel effort to produce a radiation-tolerant system that can withstand direct electron bombardment. It also describes efforts required to adapt the pixel array detector (PAD) that is commonly used in X-ray diffraction, and discusses the development of a groundbreaking prototype system based on an active pixel sensor (APS). This early implementation of an APS-based direct detection detector (DDD) has already delivered unprecedented performance in many areas exceeding the fundamental capabilities of CCD-based systems.

Publisher Summary This chapter describes the use of energy-filtered electron microscopy (EM) to enhance contrast and reduce the chromatic aberration in the imaging of thick, selectively stained specimens. For thick specimens, the resolution of electron microscopy is severely limited by chromatic aberration that results from the inability of current electron lenses to deal uniformly with beam electrons that have a distribution of energies as a result of inelastic scattering in the samples. The chapter describes a technique of automated, most-probable loss (MPL) tomography. It shows that for thick, selectively stained biological specimens, this method produces a dramatic increase in the resolution of the projected images. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. In addition, MPL tomography effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by improving resolution relative to unfiltered transmission electron microscopy (TEM).

A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.

There is an urgent need to replace film and CCD cameras as recording instruments for transmission electron microscopy (TEM). Film is too cumbersome to process and CCD cameras have low resolution, marginal to poor signal-to-noise ratio for single electron detection and high spatial distortion. To find a replacement device, we have tested a high sensitivity active pixel sensor (APS) array currently being developed for nuclear physics. The tests were done at 120 keV in a JEOL 1200 electron microscope. At this energy, each electron produced on average a signal-tonoise ratio about 20/1. The spatial resolution was also excellent with the full width at half maximum (FWHM) about 20 microns. Since it is very radiation tolerant and has almost no spatial distortion, the above tests showed that a high sensitivity CMOS APS array holds great promise as a direct detection device for electron microscopy.

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

into the Study of the Virus Life Cycle in Cells and Alzheimer’s Disease. Anna-Clare Milazzo, Jason Lanman*, James C. Bouwer, Liang Jin, Steven T. Peltier, John E. Johnson*, Stuart Kleinfelder, Nguyen-huu Xuong, Mark H. Ellisman. National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California at San Diego, La Jolla, CA 92093 * Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 # EECS Department, University of California, Irvine, CA 92697 Contact e-mail: mark@ncmir.ucsd.edu

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.