A Technical Introduction to Transmission Electron Microscopy for Soft-Matter:Imaging, Possibilities, Choices, and Technical Developments

With a significant role in material sciences, physics, (soft matter) chemistry, and biology, the transmission electron microscope is one of the most widely applied structural analysis tool to date. It has the power to visualize almost everything from the micrometer to the angstrom scale. Technical developments keep opening doors to new fields of research by improving aspects such as sample preservation, detector performance, computational power, and workflow automation. For more than half a century, and continuing into the future, electron microscopy has been, and is, a cornerstone methodology in science. Herein, the technical considerations of imaging with electrons in terms of optics, technology, samples and processing, and targeted soft materials are summarized. Furthermore, recent advances and their potential for application to soft matter chemistry are highlighted

Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction

An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins greater than 150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a 'resolution revolution', owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM

Three-dimensional imaging of bone scaffolds seeded with stem cells

W pierwszym etapie niniejszej pracy wytworzono i zasiedlono agregatami komórek macierzystych innowacyjne biomateriały. Rusztowania wykonano z mieszaniny poli(3-hydroksymaślanu-ko-3-hydroksywalerianu) (PHBV), poli(L-laktydu-ko-glikolidu) (PLGA) oraz trójfosforanu wapnia (TCP). W drugim etapie pracy, scharakteryzowano wytworzone biomateriały wykorzystując techniki rentgenowskiej mikrotomografii komputerowej z użyciem środka kontrastującego. Poddano je także analizie powierzchniowej przy pomocy mikroskopu sił atomowych. Wyniki odniesiono do materiału referencyjnego, którym były rusztowania wykonane z PHBV i PLGA. Mikrotomografia komputerowa zapewniła wnikliwą ocenę struktury rusztowań kostnych. Oprócz obrazowania komórek macierzystych umożliwiła obserwację mikrostruktury w całej objętości badanego materiału w 3D. Kolejną techniką szeroko stosowaną do badań rusztowań polimerowych była mikroskopia sił atomowych. Umożliwiła ona wizualną ocenę topografii badanych materiałów oraz analizę ich chropowatości. Wytworzone rusztowania kostne miały odpowiednie parametry dla proliferacji komórek macierzystych. Wyniki niniejszej pracy wykazały, że tomografia komputerowa jest odpowiednim narzędziem do obrazowania komórek macierzystych zasiedlonych na porowatych biomateriałach. Dodatkowo stwierdzono, że specjalnie modyfikowane sondy skanujące umożliwiły dokładniejszy w stosunku do standardowych pomiar chropowatości powierzchni rusztowań.The first stage of this study involved the preparation and seeding of innovative biomaterials with stem cell Scaffolds through a mixture of poly (3-hydroxybutyrate-co-3-hydroksy-valerat) (PHBV), poly (L-lactide-co-glycolide) (PLGA) and tricalcium phosphate (TCP). In the second stage, biomaterials were characterized using a X-ray computed microtomography (CT) with a contrast agent. They were also subjected to surface analysis using atomic force microscopy. The results were compared to the reference material, of a PHBV/PLGA composite scaffold. The computed microtomography ensured rigorous assessment of the bone scaffold structure. Apart from stem cell imaging it also enabled the observation of the microstructure in the entire volume of the material in 3D. Another technique used to study polymeric scaffold was atomic force microscopy (AFM). It allowed for the visual assessment of the topography of the tested materials, as well as the analysis of their surface roughness. The tested bone scaffolds showed appropriate parameters for stem cell proliferation. The results of this study indicated that tomography is a suitable tool for the imaging of stem cell seeding on porous biomaterials. In addition, we couclude that specially modified scanning probes enabled more accurate surface roughness measurements

In vivo characterization of the novel ebolavirus Bombali virus suggests a low pathogenic potential for humans

ABSTRACTEbolaviruses cause outbreaks of haemorrhagic fever in Central and West Africa. Some members of this genus such as Ebola virus (EBOV) are highly pathogenic, with case fatality rates of up to 90%, whereas others such as Reston virus (RESTV) are apathogenic for humans. Bombali virus (BOMV) is a novel ebolavirus for which complete genome sequences were recently found in free-tailed bats, although no infectious virus could be isolated. Its pathogenic potential for humans is unknown. To address this question, we first determined whether proteins encoded by the available BOMV sequence found in Chaerephon pumilus were functional in in vitro assays. The correction of an apparent sequencing error in the glycoprotein based on these data then allowed us to generate infectious BOMV using reverse genetics and characterize its infection of human cells. Furthermore, we used HLA-A2-transgenic, NOD-scid-IL-2γ receptor-knockout (NSG-A2) mice reconstituted with human haematopoiesis as a model to evaluate the pathogenicity of BOMV in vivo in a human-like immune environment. These data demonstrate that not only does BOMV show a slower growth rate than EBOV in vitro, but it also shows low pathogenicity in humanized mice, comparable to previous studies using RESTV. Taken together, these findings suggest a low pathogenic potential of BOMV for humans