TOM software toolbox: acquisition and analysis for electron tomography

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.electron | cryotomography | platelet | microtubule | cancer

Section: Discussionmentioning

confidence: 99%

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Wang

Stone

Kaelber

et al. 2015

“…Images were recorded with defocus values ranging from −6 to −8 μm. The projection images were aligned using cross-correlation procedures and reconstructed by a weighted back-projection algorithm as implemented in the EM and TOM software packages (31,32). Visualization and surface rendering was performed using the Amira software (33).…”

Section: Methodsmentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

Self Cite

399

306

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

“…In projections exhibiting sufficient contrast, we determined the contrast transfer function (43) and corrected for it to minimize optical aberrations introduced by the microscope. Images were processed with MatLab7 (Math Works) incorporating the TOM toolbox (44).…”

Section: Methodsmentioning

confidence: 99%

Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure

Hoffmann

Leis

Niederweis

et al. 2008

Self Cite

534

472

The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergenttreated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Gué rin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.bacterial cell wall ͉ Corynebacterium glutamicum ͉ Mycobacterium bovis ͉ Mycobacterium smegmatis ͉ mycolic acid layer M ycobacteria have evolved a complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size (up to 90 carbon atoms), a variety of extractable lipids, and pore-forming proteins (1-3). The cell wall provides an extraordinarily efficient permeability barrier to noxious compounds and contributes to the high intrinsic resistance of mycobacteria to many drugs (4). Because of the paramount medical importance of Mycobacterium tuberculosis, the ultrastructure of mycobacterial cell envelopes has been intensively studied during recent decades. The current view of the cell wall architecture is essentially based on a model suggested by Minnikin (5). He proposed that the covalently bound mycolic acids form the inner leaflet of an asymmetrical bilayer. Other lipids extractable by organic solvents were thought to form the outer leaflet, either intercalating with the mycolates (5, 6) or forming a more clearly defined interlayer plane (7). Elegant x-ray diffraction studies proved that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (8). Furthermore, freeze-fracture studies showed a second fracture plane in electron micrographs (9), indicating the existence of a hydrophobic bilayer structure external to that of the cytoplasmic membrane. Mutants or treatments affecting mycolic acid biosynthesis and the production of extractable lipids resulted in an increase of cell wall permeability in various mycobacteria and related microorganisms (10-12) and a drastic decrease of virulence, underlining the importance of the integrity of the cell wall for intracellular survival...