Roger Albert Wepf

The University of Queensland | UQ · Centre for Microscopy and Microanalysis

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre 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 st...

Cryo-ET combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. 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...

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 la...

Development of high-performance rechargeable batteries is hinged on a clear mechanistic understanding of the fundamental electrochemical processes. In operando transmission electron microscopy (TEM) provides a nanoscale insight into the dynamic microstructural evolution of battery components. The open-cell and closed-cell are the main configuration...

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 demonst...

To develop the state‐of‐the‐art polymer membrane fuel cells. both maximization of oxygen reduction reaction (ORR) activity on Pt cathode and minimization of Pt content in the cathode are required. For this challenge, the defect interface on oxide support was modified by proton beam irradiation method. Pt‐CeOx nanowire/C (Pt/C=0.02) was fabricated u...

Correlative UHV-Cryo Transfer Suite: Connecting Atom Probe, SEM-FIB, Transmission Electron Microscopy via an Environmentally-Controlled Glovebox - Volume 25 Supplement - Julie M. Cairney, Ingrid McCarroll, Yi-Sheng Chen, Katja Eder, Takanori Sato, Zongwen Liu, Alexander Rosenthal, Roger Wepf

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 pres...

Scanning electron microscopy (SEM) stereo pair and SEM photogrammetry are successful techniques for visualizing the topography of beam resistant samples. The inverse Radon transform can be used to recover an unknown three‐dimensional function from a series of parallel line integrals. The simplest way to collect a dataset suitable for three‐dimensio...

This chapter outlines the requirements to investigate macromolecular complexes at highest possible resolution by cryo‐scanning electron microscopy (cryo‐SEM). It covers the preservation of the native state, exposure of the region‐of‐ interest, the avoidance of artefacts as well as signal enhancement by metal coating, to guide through common obstacl...

Rechargeable lithium–sulfur batteries (LSBs) are of great interest in the field of energy storage due to their favorable operational characteristics, for example, high theoretical energy density and low cost. However, LSBs are limited by long‐term degradation, caused by the dissolution of intermediate lithium polysulfide phases. Further improvement...

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-F...

For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called “tOMography Nano crYo (OMNY),” dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accu...

Today ptychographic tomography in the hard x-ray regime, i.e. at energies above about 2 keV, is a well-established measurement technique. At the Paul Scherrer Institut, currently two instruments are available: one is measuring at room temperature and atmospheric pressure, and the other, so-called OMNY instrument (tOMography Nano crYo), is operating...

Enabling Atom Probe Analyses of New Materials Classes with Vacuum-Cryo-Transfer Capabilities - Volume 23 Issue S1 - Stephan S.A. Gerstl, Sebastian Tacke, Y-S. Chen, Julia Wagner, Roger Wepf

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 i...

Heavy hydrogen gets frozen in place Hydrogen embrittlement contributes to the failure of steel in a wide variety of everyday applications. Various strategies to mitigate hydrogen embrittlement, such as adding carbides into the steel, are hard to validate because we are unable to map the hydrogen atoms. Chen et al. combined fluxing steel samples wit...

The conservation of the native state during sample preparation is mandatory for a correct interpretation of any micrograph. Particularly for EM, the preservation of the pristine architecture is challenging. If not imaged in situ (1), two different strategies can be followed to prepare hydrated samples for electron microscopy: the conventional and t...

Ionic liquids are low-melting salts which are fluid at room temperature. They show a very low vapor pressure and are therefore persisting as liquids under the high vacuum conditions in an electron microscope. They are electrical conductive as well. The ionic liquids tested are mixable with water, thus solutions can be easily diluted to suitable vis...

The preservation of the native sample state during manipulation and exchange between sample preparation and imaging devices is a prerequisite for artefact and contamination free structure investigation. In particular during cryo-transfer and (re-)mounting of frozen hydrated samples, which often in electron microscopy (EM), have a very large surface...

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-vacuu...

After publication of the article it has been notified to the authors that the organelles proposed to be liquid droplets are most probably polyphosphate bodies, which would explain the high density measured for these organelles in the frozen hydrated specimen. Polyphosphate bodies are known to be very electron-dense, as measured in 2D projections of...

We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180nm. 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...

In its infancy, atom probe microscopy began as an analytical instrument primarily for metals and later semiconductors. As laser pulsing technology rapidly improved and computational capabilities were integrated, expediting many functions, analysis of additional materials classes were enabled, such as oxides, ceramics, and minerals [1]. All of these...

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...

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, part...

Email of the presenting author: annegreet.bittermann@scopem.ethz.ch Free hemoglobin (Hb) forms under oxidizing conditions large protein aggregates, which are heavily and actively taken up by endothelial cells: Membrane ruffles reach out to grab these particles. Several membrane lamella try to wrap them independently and pull them into the cytoplasm...

Email of the presenting author: alex.demarco@fei.com Correlative light and electron microscopy (CLEM) aims at combining the large field of view and chemical specificity of fluorescence microscopy with the high resolution ultra-structural details revealed by electron microscopy. CLEM can be extremely powerful in extending electron microscopy analysi...

Keywords: Enter up to three keywords Correlative light and electron microscopy (CLEM) is a powerful tool in life science to combine large-scale volume imaging of cells or tissues by LM with a high-resolution description of their morphology using EM. The combination of 3D microscopy techniques such as CLSM with FIB-SEM or serial block face SEM (SBF-...

Correlative light and electron microscopy (CLEM) is a powerful tool in life science to combine large-scale volume imaging of cells or tissues by LM with a high-resolution description of their morphology using EM. The combination of 3D microscopy techniques such as CLSM with FIB-SEM, serial block face SEM (SBF-SEM) [1] or array tomography (AT) [2] o...

Three-dimensional scanning electron microscopy generates quantitative volumetric structural data from SEM images of macromolecules. This technique provides a quick and easy way to define the quaternary structure and handedness of protein complexes. Here, we apply a variety of preparation and imaging methods to filamentous actin in order to explore...

Please create your abstract using the following template and send this word document to biobarriers2014@mx.uni-­‐saarland.de NOTE: Please save your abstract file according to the name of the presenting author as a 'doc' file (Author A B.doc). Abstract limit is 1 page A4 (Biographical sketch and contact info not included) Modern tissue biology deals...

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, part...

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are...

Ultrastructure investigations in biology are to date dominated by TEM. However, modern field emission SEM resolves structural details of biological specimen in the range of 1-2 nm. Thus, SEM has the lateral resolution power to image the compartment forming structural element of cells: lipid membranes! Focused ion beam-SEM has emerged as one of the...

of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The ion conductivity of two series of porous ceramic diaphragms impregnated with caustic potash was investigated by electrochemical impedance spectroscopy. To understand the impact of the pore structure on ion conductivity, the three-dimensional (3-D) pore geometry of the diaphragms was characterized with synchrotron x-ray absorption tomography. Io...

Although the presence of phase-contrast information in bright field images recorded with a scanning transmission electron microscope (STEM) has been known for a long time, its systematic exploitation for the structural characterization of materials began only with the availability of aberration-corrected microscopes that allow sufficiently large il...

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 experime...

A new method is described that combines a microfluidic continuous-flow reactor for the controlled formation of liposomes with instantaneous immobilization by means of ultra-rapid 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 bef...

HET-S in the presence of HET-s(218–289) amyloid seeds makes holes in liposomes observed by freeze-fracture electron microscopy while HET-S alone does not. (A) Cryo-SEM (scanning electron microscopy) images of freeze fractures of 100 nm diameter extruded liposomes incubated in the presence of a mixture of HET-S and HET-s(218–289) fibril seeds. These...

TM helix predictions (output of TMHMM with residues 1–33 as input) are shown for several interconverting variants of the TM region. The genotype of each construct is in italics and its phenotype is in square brackets. The histograms are the per-residue probability output from TMHMM with the [Het-S] phenotypical proteins in grey and the location of...

Detailed discussion on the discrepancies between in vivo and in vitro HET-S activity. (PDF)

Aliphatic region of the DARR solid-state NMR spectrum (100 ms mixing) of HET-S aggregates (blue) and HET-s(218–289) fibrils (black contours). (PDF)

Sequence alignment of HET-s and HET-S. The 13 out of 289 not identical residues are spread over the whole protein and are highlighted in red. Both proteins consist of a globular N-terminal HeLo domain (residues 1–227) and a C-terminal PFD (residues 218–289, underlined in black). The secondary structural elements are indicated for the HeLo domain as...

HET-S/s variant phenotype correlation to calcein leakage and TM prediction. (PDF)

Experimental parameters of the solid-state NMR spectra. Their pulse sequences are given in [26]. The DREAM transfer is described in [59]. (PDF)

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 prote...

While HRTEM is the well-established method to characterize the structure of dodecagonal tantalum (vanadium) telluride quasicrystals and their periodic approximants, phase-contrast imaging performed on an aberration-corrected scanning transmission electron microscope (STEM) represents a favorable alternative. The (Ta,V)151Te74 clusters, the basic st...

FIB/SEM allows targeted 3D imaging of cellular fine structure at high resolution on a small volume. While it is possible to perform FIB cross sections on critical-point dried or freeze dried cells, fine structure detail is better preserved if the cellular water is not just removed but replaced by organic resin compounds. Therefore biological sample...

Besides the planktonic way of living, microbes are able to adhere to surfaces or interfaces and to form organized communities, so-called biofilms, which are embedded in a matrix of extracellular polymeric substances that they have produced. These communities are beneficial to humans as microflora colonising surfaces of the human body. They also for...

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-d...

Ultrastructure investigations in biology are to date dominated by TEM. However, modern field emission SEM resolves structural details of biological specimen in the range of 1-2 nm. Thus, SEM has the lateral resolution power to image the compartment forming structural element of cells: lipid membranes! Focused ion beam-SEM has emerged as one of the...

Ultrastructure investigations in biology are to date dominated by TEM. However, modern field emission SEM resolves structural details of biological specimen in the range of 1-2 nm. Thus, SEM has the lateral resolution power to image the compartment forming structural ele-ment of cells: lipid membranes! Focused ion beam-SEM has emerged as one of the...

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 co...

The width, w, and height, h, of dendronized polymers (DPs) adsorbed onto mica and highly oriented pyrolitic graphite (HOPG) were characterized by atomic force microscopy (AFM) and electron microscopy (EM). The study utilized a homologous series of generations g = 1-5, hence enabling coadsotption and characterization under identical conditions and t...

Supplementary Figures S1-S9, Supplementary Tables S1-S3, Supplementary Notes 1-4 and Supplementary References

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...

We present a comprehensive study on the influence of Ar(+) ion milling parameters in the range of low acceleration voltages (0.5-6 kV) and etching angles (3-10(°)) on the quality of standard high resolution Si TEM samples. The quality was assessed by the evaluation of HR-TEM images acquired from real TEM samples considering the thickness of the amo...

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...

Clay-rich sedimentary rocks are considered as seal lithologies for hosting radioactive waste or as caprocks for geological carbon sequestration sites. Evaluation of the rock's sealing capacity requires a comprehensive understanding of mass transport processes, which, in turn, demands knowledge of the D structure of pore space. Here, the use of focu...