TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

<p>Abstract</p> <p>Background</p> <p>Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.</p> <p>Results</p> <p>TomoJ provides a user-friendly interface for alignment, reconstruction, and combination of multiple tomographic volumes and includes the most recent algorithms for volume reconstructions used in three-dimensional electron microscopy (the algebraic reconstruction technique and simultaneous iterative reconstruction technique) as well as the commonly used approach of weighted back-projection.</p> <p>Conclusion</p> <p>The software presented in this work is specifically designed for electron tomography. It has been written in Java as a plug-in for ImageJ and is distributed as freeware.</p

Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

Investigation of materials for catalysis with electron tomography

Elektronentomographie mit dem Transmissionselektronenmikroskop (TEM) ermöglicht die Erstellung dreidimensionaler Darstellungen (Tomogramme) von Proben in der Größenordnung von einigen Nanometern bis hin zu einigen Mikrometern. Im Rahmen dieser Arbeit wurden verschiedene auf Ruthenium basierende Werkstoffe für die Katalyse in Brennstoffzellen untersucht. Die Tomographie liefert, im Gegensatz zu gewöhnlichen TEM Bildern (Projektionen), Aufschluss über die Verteilung und Erreichbarkeit der Katalysatorpartikel auf bzw. in dem Trägermaterial. Es konnte gezeigt werden, dass neben qualitativen Vergleichen der Verteilung der Rutheniumpartikel auf/in dem Kohleträgermaterial verschieden hergestellter Proben auch detaillierte quantitative Analysen möglich sind. Da die Katalyse an heterogenen Katalysatoren an der Oberfläche des Katalysators stattfindet, spielen neben der Größe der Oberfläche auch die unterschiedlichen Koordinationszahlen verschieden orientierter Facetten der Katalysatorpartikel eine Rolle. Dazu wurde erstmalig ein Algorithmus entwickelt, der es erlaubt, viele verschiedene Partikel in dreidimensionalen Datensätzen automatisch hinsichtlich Facettierung zu analysieren. Durch die teilweise Einbettung der Katalysatorpartikel in das Trägermaterial ist eine Unterscheidung der bedeckten und unbedeckten Oberfläche nötig, da nur der unbedeckte Teil der Katalysatoroberfläche von den Reaktanten erreicht werden kann. Neben dieser unbedeckten Oberfläche ist durch die teilweise Einbettung auch die Ausrichtung der Katalysatorpartikel in Bezug zur lokalen Oberfläche des Trägers bedeutsam, da so statistische Untersuchungen der unbedeckten Facettentypen möglich werden. Zu den durchgeführten Charakterisierungen wie: Partikelverteilung innerhalb des Trägers, Größenverteilung, Oberflächen, Volumina, Formanalyse und der lokalen Ausrichtung, wurden Erkenntnisse gewonnen, die es erlauben, den untersuchten Katalysatortyp während der Herstellung weiter zu optimieren. Es konnte zudem gezeigt werden, dass die entwickelten Bildanalysemethoden sich auch auf tomographische Datensätze anderer Messmethoden wie z.B. Neutronen- und Focused Ion Beam-Tomographie anwenden lassen.Electron tomography with a transmission electron microscope (TEM) enables creation of three-dimensional representations (tomograms) of samples in the range of a few nanometres to a few micrometres. In the frame of this thesis different ruthenium-based materials for catalysis in fuel cells were investigated. Tomography, in contrast to common TEM images (projections), yields information about the distribution and accessibility of the catalyst particles on or in the support material. It was shown that in addition to qualitative comparisons of the distribution of ruthenium particles on/in the carbon support material of differently manufactured samples, quantitative analyses are also possible. Since catalysis on heterogeneous catalysts takes place at the surface of the catalyst, the amount of surface area matters as do the coordination numbers of differently oriented facets of the catalyst particles. For this purpose a new algorithm was developed that allows to automatically analyse faceting of many different particles in a three-dimensional dataset. Due to the partial embedding of the catalyst particles into the support material only the uncovered fraction of the catalyst surface is accessible to the reactants and therefore a differentiation between the covered and uncovered catalyst surface is necessary. Apart from this uncovered surface, the orientation of the catalyst particles relative to the local support surface is also important since this allows statistical investigation of the uncovered facet types. In addition to the conducted characterizations such as: particle distribution within the support, size distribution, surface areas, volumes, shape analysis and the local orientation, new insights were gained which allow optimization of the examined catalyst during production. Furthermore, it could be shown that the developed image analysis methods can be applied to tomographic datasets from other measurement techniques such as neutron and focused ion beam tomography

3-Dimensional Electron Microscopy of Biological Specimens

Three-dimensional (3D) imaging is an important tool in electron microscopy, especially in biological specimens where the main focus is the structure of the cells. Many times important information is lost because the exact orientation of a specimen is unknown. We tested two different 3D imaging techniques, focused ion beam (FIB) slice and view, and cryo-FIB thinning of samples for use in cryo transmission electron tomography (cryo-TEM) and cryo-electron tomography (cryo-ET). We began our research with room temperature FIB slice and view, with an intention to move onto slice and view at cryogenic temperatures. We found this technique to be difficult to control and the time required to produce results was simply too high. We moved on to investigating cryo-FIB milling as a tool for thinning cryo-ET specimens. Advances in cryo-ET have enabled high-resolution 3D imaging of complex assemblies and determination of cellular architectures in their close-to-native states. However, one major limitation, the accessible specimen thickness, has hindered its broader application in cellular biology. Recent efforts have been made to create thin, frozen-hydrated sections using cryo-ultramicrotomy, but with many mechanical artifacts and low yields. Here, we report a method that applies a focused ion beam (FIB) at cryogenic temperature (cryo-FIB) to reduce the thickness of frozen-hydrated cells, including mammalian cells, to a degree suitable for cryo-ET

Electron Cryotomography

Electron cryotomography (ECT) is an emerging technology that allows thin samples such as macromolecular complexes and small bacterial cells to be imaged in 3-D in a nearly native state to “molecular” (~4 nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal filaments, cell wall elements, motility machines, chemoreceptor arrays, internal compartments, and other ultrastructures. This article describes the technique and summarizes its contributions to bacterial cell biology. For comparable recent reviews, see (Subramaniam 2005; Jensen and Briegel 2007; Murphy and Jensen 2007; Li and Jensen 2009). For reviews on the history, technical details, and broader application of electron tomography in general, see for example (Subramaniam and Milne 2004; Lucić et al. 2005; Leis et al. 2008; Midgley and Dunin-Borkowski 2009)

Cryoelectron Tomography of Bacteria and their Macromolecular Machines

Cryoelectron tomography (CET) fills a glaring gap in the imaging capabilities of biology by reconstructing cells to medium resolution. The technique was applied in three areas to understand biology’s macromolecular machines: (1) the quaternary structure of the octahedrally-cored E. coli pyruvate dehydrogenase (PDHC) and 2-oxoglutarate dehydrogenase (OGDHC) complexes in vitro; (2) the ultrastructure of the spirochete Treponema primitia; and (3) the structure of the in situ flagellar motors from T. primitia, Hylemonella gracilis, Caulobacter crescentus, and Vibrio cholerae. Whereas the complexes PDHC and OGDHC were thought to have their subunit proteins E1 and E3 bound directly to the octahedral E2 core—the so-called face/edge model—it was discovered that the subunits are flexibly tethered 11 nm from the corners of the core. Several novel structures were discovered in the spirochete T. primitia. Spirochetes are spiral-shaped cells that propel themselves with periplasmic, not external, flagella. Bowl-shaped structures dot its surface and hook-like appendages that form arcades stripe the length of the cell. Fibrils extend from its cell tips that might help attach the cells to surfaces. Inside the periplasm, porous, cone-shaped structures reside at each cell tip and a second periplasmic layer undergirds its outer membrane, which might prevent the periplasmic flagella from rupturing the cell. Previous imaging of the flagellar motor produced either high-resolution reconstructions of the purified basal body removed from its context or low-resolution images of the in situ motor. Our in situ 3-D reconstructions described for the first time the structure of the stators, the membrane embedded component that spins the rotor. Novel shapes were discovered that indicate there are various attachments and versions of the flagellar motor that were never expected