A cryogenic, coincident fluorescence, electron, and ion beam microscope.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Active Pt-Nanocoated Layer with Pt-O-Ce Bonds on a CeO x Nanowire Cathode Formed by Electron Beam Irradiation.

A Pt-nanocoated layer (thickness of approx. 10-20 nm) with Pt-O-Ce bonds was created through the water radiolysis reaction on a CeO x nanowire (NW), which was induced by electron beam irradiation to the mixed suspension of K2PtCl4 aqueous solution and the CeO x NW. In turn, when Pt-nanocoated CeO x NW/C (Pt/C ratio = 0.2) was used in the cathode layer of a membrane electrode assembly (MEA), both an improved fuel cell performance and stability were achieved. The fuel cell performance observed for the MEA using Pt-nanocoated CeO x NW/C with Pt-O-Ce bonds, which was prepared using the electron beam irradiation method, improved and maintained its performance (observed cell potential of approximately 0.8 V at 100 mW cm-2) from 30 to 140 h after the start of operation. In addition, the activation overpotential at 100 mA cm-2 (0.17 V) obtained for MEA using Pt-nanocoated CeO x NW/C was approximately half of the value at 100 mA cm-2 (0.35 V) of MEA using a standard Pt/C cathode. In contrast, the fuel cell performance (0.775 V at 100 mW cm-2 after 80 h of operation) of MEA using a nanosized Pt-loaded CeO x NW (Pt/C = 0.2), which was prepared using the conventional chemical reduction method, was lower than that of MEA using a Pt-nanocoated CeO x /C cathode and showed reduction after 80 h of operation. It is considered why the nanocoated layer having Pt-O-Ce bonds heterogeneously formed on the surface of the CeO x NW and the bare CeO2 surface consisting of Ce4+ cations would become unstable in an acidic atmosphere. Furthermore, when a conventional low-amount Pt/C cathode (Pt/C = 0.04) was used as the cathode layer of the MEA, its stable performance could not be measured after 80 h of operation as a result of flooding caused by a lowering of electrocatalytic activity on the Pt/C cathode in the MEA. In contrast, a low-amount Pt-nanocoated CeO x NW (Pt/C = 0.04) could maintain a low activation overpotential (0.22 V at 100 mA cm-2) of MEA at the same operation time. Our surface first-principles modeling indicates that the high quality and stable performance observed for the Pt-nanocoated CeO x NW cathode of MEA can be attributed to the formation of a homogeneous electric double layer on the sample. Since the MEA performance can be improved by examining a more effective method of electron beam irradiation to all surfaces of the sample, the present work result shows the usefulness of the electron beam irradiation method in preparing active surfaces. In addition, the quantum beam technology such as the electron beam irradiation method was shown to be useful for increasing both performance and stability of fuel cells.

Cryo-ultramicrotomy and Mass Spectrometry Imaging Analysis of Nudibranch Microstructures.

In this paper, we investigate the presence of latrunculin A in the outer rim of a nudibranch Chromodoris kuiteri and show that by combining ultrathin cryosection methods with MALDI MSI we can achieve improved lateral (x and y) resolution and very high resolution in the z dimension by virtue of the ultrathin 200 nm thin cryosections. We also demonstrate that a post ionization laser increases sensitivity. Recent advances in MALDI source design have improved the lateral resolution (x and y) and sensitivity during MSI. Taken together, very high z resolution, from ultrathin sections, and improved lateral (x and y) resolution will allow for subcellular molecular imaging with the potential for subcellular 3D volume reconstruction.

Can one determine the density of an individual synthetic macromolecule?

Dendronized polymers (DPs) are large and compact main-chain linear polymers with a cylindrical shape and cross-sectional diameters of up to ∼15 nm. They are therefore considered molecular objects, and it was of interest whether given their experimentally accessible, well-defined dimensions, the density of individual DPs could be determined. We present measurements on individual, deposited DP chains, providing molecular dimensions from scanning and transmission electron microscopy and mass-per-length values from quantitative scanning transmission electron microscopy. These results are compared with density values obtained from small-angle X-ray scattering on annealed bulk specimen and with classical envelope density measurements, obtained using hydrostatic weighing or a density gradient column. The samples investigated comprise a series of DPs with side groups of dendritic generations g = 1-8. The key findings are a very large spread of the density values over all samples and methods, and a consistent increase of densities with g over all methods. While this work highlights the advantages and limitations of the applied methods, it does not provide a conclusive answer to the question of which method(s) to use for the determination of densities of individual molecular objects. We are nevertheless confident that these first attempts to answer this challenging question will stimulate more research into this important aspect of polymer and soft matter science.

Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography.

Electron cryotomography is able to visualize macromolecular complexes in their cellular context, in a frozen-hydrated state, and in three dimensions. The method, however, is limited to relatively thin samples. Cryo-focused ion beam (FIB) milling is emerging as a powerful technique for sample thinning prior to cryotomography imaging. Previous cryo-FIB milling reports utilized custom-built non-standard equipment. Here we present a workflow and the required commercially available instrumentation to either implement the method de novo, or as an upgrade of pre-existing dual beam milling instruments. We introduce two alternative protocols and the respective sample holders for milling. The "bare grid holder" allows for milling on plain grids, having the advantage of enabling relatively shallow milling angles for wedge geometries. The "Autogrid holder" is designed for milling grids clamped into a mechanical support ring (Autogrid), resulting in increased stability for lamella geometries. We applied the workflow to prepare samples and record high-quality tomograms of diverse model organisms, including infected and uninfected HeLa cells, amoebae, yeast, multicellular cyanobacteria, Pseudomonas aeruginosa and Escherichia coli cells. The workflow will contribute to the dissemination of electron cryotomography of cryo-FIB milled samples in the biological sciences.

Correlation of live-cell imaging with volume scanning electron microscopy.

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi µ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods.

A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics.

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.

Three-dimensional mass density mapping of cellular ultrastructure by ptychographic X-ray nanotomography.

We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 µm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition.

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Correlative 3D imaging: CLSM and FIB-SEM tomography using high-pressure frozen, freeze-substituted biological samples.

Correlative light and electron microscopy aims at combining data from different imaging modalities, ideally from the same area of the one sample, in order to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern 3D imaging techniques opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale. Here we present an approach to correlate 3D light microscopy data with volume data from focused ion beam-scanning electron microscopy. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en bloc imaging, is described. It is based on including fluorescent labeling during freeze-substitution, which enables histological context description of the structure of interest by confocal laser scanning microscopy prior to high-resolution electron microscopy. This information can be employed to relocate the respective structure in the electron microscope. This approach is most suitable for targeted small 3D volume correlation and has the potential to extract statistically relevant data of structural details for systems biology.

Simultaneous correlative scanning electron and high-NA fluorescence microscopy.

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Freezing continuous-flow self-assembly in a microfluidic device: toward imaging of liposome formation.

A new method is described that combines a microfluidic device for the controlled formation of liposomes with instantaneous immobilization by means of ultrarapid cooling. The microfluidic device is composed of capillaries to hydrodynamically focus a stream of lipids dissolved in 2-propanol by two adjacent aqueous buffer streams before rapidly cooling by propane jet-freezing. The capillary containing the frozen sheath-flow is subsequently separated from the flow-focusing unit and trimmed with cryo-ultramicrotomy for imaging with cryo-scanning electron microscopy (SEM). The emergence of liposomes could be visualized by cryo-SEM without the need for chemical fixation or labeling. We demonstrate that the method is capable of revealing in more detail the formation of nonequilibrium liposomes. Partially and completely formed liposomes were observed at the miscible alcohol-buffer interface. The number density of lipid vesicles varied along the focused interface, and we frequently found clusters of liposomes. Additionally, evidence for the formation of disclike transient intermediates is presented. The method is not limited to studying self-assembly processes only. It can be extended to other biochemical reactions, crystallization processes, and even systematic interfacial mixing studies between different solvents.

Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

The mechanism of toxicity in HET-S/HET-s prion incompatibility.

The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the ß-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.

Phase tomography from x-ray coherent diffractive imaging projections.

Coherent diffractive imaging provides accurate phase projections that can be tomographically combined to yield detailed quantitative 3D reconstructions with a resolution that is not limited by imaging optics. We present robust algorithms for post-processing and alignment of these tomographic phase projections. A simple method to remove undesired constant and linear phase terms on the reconstructions is given. Also, we provide an algorithm for automatic alignment of projections that has good performance even for samples with no fiducial markers. Currently applied to phase projections, this alignment algorithm has proven to be robust and should also be useful for lens-based tomography techniques that pursue nanoscale 3D imaging. Lastly, we provide a method for tomographic reconstruction that works on phase projections that are known modulo 2π, such that the phase unwrapping step is avoided. We demonstrate the performance of these algorithms by 3D imaging of bacteria population in legume root-nodule cells.

Measuring single-nanoparticle wetting properties by freeze-fracture shadow-casting cryo-scanning electron microscopy.

Nanoparticles at fluid interfaces are central to a rapidly increasing range of cutting-edge applications, including drug delivery, uptake through biological membranes, emulsion stabilization and the fabrication of nanocomposites. Understanding nanoscale wetting is a challenging issue, still unresolved for individual nanoparticles, and is essential in designing nanoparticle-building blocks with controlled surface properties. The core information about the structural and thermodynamic properties of particles at fluid interfaces is enclosed in the three-phase contact angle θ. Here we present a novel in situ method, on the basis of freeze-fracture shadow-casting cryo-scanning electron microscopy, that allows the measurement of contact angles of individual nanoparticles with 10 nm diameter, and thus greatly surpasses the current state of the art. We study hydrophilic and hydrophobic, organic and inorganic nanoparticles, demonstrating general applicability to systems of fundamental and applied nanotechnological interest. Significant heterogeneity in the wetting of nanoparticles is observed.

Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by electron cryo-microscopy.

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' ß-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.

Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuno-canalicular network.

Up to now, a quantitative three-dimensional (3D) assessment of the lacuno-canalicular network (LCN) within bone has not been achieved in a comprehensive way and the LCN has mostly been investigated using two-dimensional imaging methods only. First attempts for the 3D assessment of the osteocytes and their cell processes have been reported using different imaging techniques. Nevertheless, various experimental limitations allowed for assessment of isolated or incompletely interconnected osteocytes only. On the other hand, serial focused ion beam/scanning electron microscopy (FIB/SEM) currently seems to be a promising imaging method for quantitative 3D assessment of the LCN. However, combined 3D visualization and quantification of the LCN using serial FIB/SEM imaging has not been reported so far. The aim of this study was to provide a proof of concept that serial FIB/SEM meets all requirements for quantitative 3D imaging of the LCN. To this end, we developed a new bone sample preparation protocol for serial FIB/SEM imaging providing a resolution on the order of 30nm. This technique was successfully applied to the mid-diaphysis of a mouse femur. Moreover, we devised and applied novel measures for subsequent quantitative 3D morphometry of the LCN. Briefly, serial FIB/SEM was shown to be an appropriate technique to quantify the morphology of the LCN truly in 3D. This will allow investigating bone matrix changes on an ultrastructural level, which result from aging, disease, and treatment.

Ptychographic X-ray computed tomography at the nanoscale.

X-ray tomography is an invaluable tool in biomedical imaging. It can deliver the three-dimensional internal structure of entire organisms as well as that of single cells, and even gives access to quantitative information, crucially important both for medical applications and for basic research. Most frequently such information is based on X-ray attenuation. Phase contrast is sometimes used for improved visibility but remains significantly harder to quantify. Here we describe an X-ray computed tomography technique that generates quantitative high-contrast three-dimensional electron density maps from phase contrast information without reverting to assumptions of a weak phase object or negligible absorption. This method uses a ptychographic coherent imaging approach to record tomographic data sets, exploiting both the high penetration power of hard X-rays and the high sensitivity of lensless imaging. As an example, we present images of a bone sample in which structures on the 100 nm length scale such as the osteocyte lacunae and the interconnective canalicular network are clearly resolved. The recovered electron density map provides a contrast high enough to estimate nanoscale bone density variations of less than one per cent. We expect this high-resolution tomography technique to provide invaluable information for both the life and materials sciences.