Roger Wepf

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 100u2009nm 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.

Towards quantitative 3D imaging of the osteocyte lacuno-canalicular network

Osteocytes are the most abundant cells in bone and the only cells embedded in the bone mineral matrix. They form an extended, three-dimensional (3D) network, whose processes interconnecting the cell bodies reside in thin canals, the canaliculi. Together with the osteocyte lacunae, the canaliculi form the lacuno-canalicular network (LCN). As the negative imprint of the cellular network within bone tissue, the LCN morphology is considered to play a central role for bone mechanosensation and mechanotransduction. However, the LCN has neither been visualized nor quantified in an adequate way up to now. On this account, this article summarizes the current state of knowledge of the LCN morphology and then reviews different imaging methods regarding the quantitative 3D assessment of bone tissue in general and of the LCN in particular. These imaging methods will provide new insights in the field of bone mechanosensation and mechanotransduction and thus, into processes of strain sensation and transduction, which are tightly associated with osteocyte viability and bone quality.

The Largest Synthetic Structure with Molecular Precision: Towards a Molecular Object

Pushing the limits: A 200A - 10 Da structurally defined, linear macromolecule (PG5) has a molar mass, cross-section dimension, and cylindrical shape that are comparable to some naturally occurring objects, such as amyloid fibrils or certain plant viruses. The macromolecule is resistant against flattening out on a surface; the picture shows PG5 embracing the tobacco mosaic virus (TMV).

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.

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.

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.

The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

A nontoxic functional prion activates toxicity in the HET-S/HET-s fungal heterokaryon incompatibility system by converting HET-S into a cytotoxic membrane protein.

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.

Direct observation of individual hydrogen atoms at trapping sites in a ferritic steel

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 with deuterium and a cryogenic transfer protocol to minimize hydrogen diffusion, allowing for detailed structural analysis (see the Perspective by Cairney). Their findings revealed hydrogen trapped in the cores of the carbide precipitates. The technique will be applicable to a wide range of problems, including corrosion, catalysis, and hydrogen storage. Science, this issue p. 1196; see also p. 1128 The combination of deuteration and a cryogenic transfer protocol reveals hydrogen locations in high-strength steel. The design of atomic-scale microstructural traps to limit the diffusion of hydrogen is one key strategy in the development of hydrogen-embrittlement–resistant materials. In the case of bearing steels, an effective trapping mechanism may be the incorporation of finely dispersed V-Mo-Nb carbides in a ferrite matrix. First, we charged a ferritic steel with deuterium by means of electrolytic loading to achieve a high hydrogen concentration. We then immobilized it in the microstructure with a cryogenic transfer protocol before atom probe tomography (APT) analysis. Using APT, we show trapping of hydrogen within the core of these carbides with quantitative composition profiles. Furthermore, with this method the experiment can be feasibly replicated in any APT-equipped laboratory by using a simple cold chain.

Cryo-FIB-nanotomography for quantitative analysis of particle structures in cement suspensions.

Cryo‐FIB‐nanotomography is a novel high‐resolution 3D‐microscopy technique, which opens new possibilities for the quantitative microstructural analysis of complex suspensions. In this paper, we describe the microstructural changes associated with dissolution and precipitation processes occurring in a fresh cement paste, which has high alumina and sulphate contents. During the first 6 min, precipitation of ettringite leads to a general decrease of the particle size distribution. In the unhydrated cement paste almost no particles smaller than 500 nm are present, whereas after 6 min this size class already represents 9 vol%. The precipitation of ettringite also leads to a significant increase of the particle number density from 0.294*109/mm3 at t0min to 20.55*109/mm3 at t6min. Correspondingly the surface area increases from 0.75 m2/g at t0min to 2.13 m2/g at t6min. The small ettringite particles tend to form agglomerates, which strongly influence the rheological properties. The particular strength of cryo‐FIB‐nt is the potential to quantify particle structures in suspension and thereby also to describe higher‐order topological features such as the particle–particle interfaces, which is important for the study of agglomeration processes.