Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Braet, Filip; Zanger, Ronald De; Wisse, Eddie

doi:10.1046/j.1365-2818.1997.1940755.x

Cited by 287 publications

(221 citation statements)

References 8 publications

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“…The surface textures of TM in the Lowicryl sections seen here are in agreement with previous studies by scanning electron microscopy of bacterial sections in which specimen-related reliefs of 5-6 nm were found near membranous organelles (Kellenberger et al, 1987). Some of the surface texture of the larger areas of the section observed by AFM at low magnification corresponds to previously published images of sections of cellular regions embedded in different polymeric resins observed using AFM (Yamamoto and Tashiro, 1994;Braet et al, 1997). Previous TEM studies of stained sections of tubular myelin have revealed that some regions of the square lattice networks are more visible than others in the micro- graphs (Gil and Reiss, 1978).…”

Section: Resultssupporting

confidence: 91%

“…A multimodal approach using techniques such as electron and atomic force microscopy on the same macromolecular complex may yield specific three-dimensional information on such systems (Lal and Proksch, 1997). Recent studies using AFM on electron microscopy sections of whole cells have shown that additional information on organelle structures which was not available using either technique by itself, can be obtained (Braet et al, 1997;Canet et al, 1996). The limitations of applying AFM imaging to mixed complex macromolecular systems lie in the somewhat arbitrary identification of the imaged structures, since the three-dimensional surface topography of biological structures may be different from the two-dimensional images obtained using techniques such as transmission electron microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…The advantage of AFM over conventional techniques, such as electron microscopy (EM), is that three-dimensional information regarding the materials can be obtained at the molecular level, without the heavy metal staining as in transmission EM or surface coating employed in scanning EM (Hansma et al, 1997). The structures of unstained cellular organelles in thin sections embedded in typical polymeric materials used for EM histology, such as Lowicryl and London Resin-White (LR-White), have been characterized (Braet et al, 1997;Yamamoto and Tashiro, 1994). In such sections, the organelles protrude out of the plane of the polymer surface, allowing the topography of such structures to be conveniently observed by AFM (Yamamoto and Tashiro, 1994).…”

Section: Introductionmentioning

confidence: 99%

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Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

Nag

Munro

Hearn

et al. 1999

Journal of Structural Biology

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Pulmonary surfactant stabilizes the lung by reducing surface tension at the air-water interface of the alveoli. Surfactant is present in the lung in a number of morphological forms, including tubular myelin (TM). TM is composed of unusual 40 ؋ 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymerembedded Lowicryl and London Resin-White (LRWhite) unstained thin sections. AFM was used in imaging regions of the sections where TM was detected by transmission electron microscopy (EM) of corresponding stained sections. Tapping-and contact-mode AFM imaging of the unstained sections containing TM indicated a highly heterogeneous surface topography with height variations ranging from 10 to 100 nm. In tapping-mode AFM, tubular myelin was seen as hemispherical protrusions of 30-70 nm in diameter, with vertical dimensions of 5-8 nm. In contact-mode AFM and with phase imaging using a sharper (G10 nm nominal radius) probe, square open-ended tubes which resembled typical electron micrographs of such regions were observed. The cross-hatch structures observed inside the tubes using EM were not observed using AFM, although certain multilobe structures and topographic heterogeneity were detected inside some tubes. Other regions of multilamellar bodies and some regions where such bilayer lamella appear to fuse with the tubes were found in association with TM using AFM. EM of acetone-delipidated tubes in LR-White revealed rectangular tubular cores containing cross-hatched structures, presumably protein skeletons. AFM surface topography of these regions showed hollow depressions at positions at which the protein was anticipated instead of the protrusions seen in the lipid-containing sections. Gold-labeled antibody to surfactant protein A was found associated somewhat randomly within the regions containing the protein skeletons. The topography of the gold particles was observed as sharp peaks in contact-mode AFM. This study suggests a method for unambiguous detection of threedimensional nanotubes present in low abundance in a biological macromolecular complex. Only limited detection of proteins and lipids in surfaces of embedded tubular myelin was possible. EM and AFM imaging of such unusual biological structures may suggest unique lipid-protein associations and arrangements in three dimensions. Academic Press

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

Nag

Munro

Hearn

et al. 1999

Journal of Structural Biology

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“…Samples were dehydrated in graded solutions of ethanol and hexamethyldisilazane (HMDS, Sigma, St. Louis, MO, USA). HMDS has been shown to be as effective at preserving biological sample structure as critical point drying (Braet et al 1997). HMDS was evaporated overnight in a fume hood, and basement membrane samples were stored under desiccant for a minimum of 3 days to ensure complete dehydration.…”

Section: Basement Membrane Isolationmentioning

confidence: 99%

Vascular growth factor binding kinetics to the endothelial cell basement membrane, with a kinetics-based correction for substrate binding

Clyne

Edelman

2009

Cytotechnology

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Vascular growth factors, including vascular endothelial growth factor and fibroblast growth factor-2, bind to heparan sulfate proteoglycans in the basement membrane. While this binding, storage, and release system provides a critical model for controlled drug release devices, basement membrane-growth factor binding kinetics have not been fully established. We modified endothelial cell-growth factor binding kinetics protocols for the basement membrane. The basement membrane showed low affinity for fibroblast growth factor-2 (K d = 185.8 nM), with a slow off rate (k off = 0.00338 min -1 ). However, results were confounded by growth factor binding to tissue culture polystyrene in a manner strikingly similar to basement membrane. Since substrate binding could not be blocked, a binding kinetics based correction technique was developed to account for polystyrene growth factor binding. This method was validated by conducting binding kinetics experiments on bacteriologic plates that exhibit little growth factor binding. This novel method will improve our understanding of cell and protein interaction with the basement membrane in health and disease. They can also further be applied to develop biomimetic drug delivery systems.

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“…Briefly, liver tissue was glutaraldehyde-prefixed, cut to 1 mm 3 samples and following glutaraldehyde fixation, tissue blocks were postfixed in 1% osmium tetroxide and dehydrated in graded ethanol solutions. The liver tissue was then dried with hexamethyldisilazane and subsequently broken in liquid nitrogen, mounted on stubs and sputter coated with a thin layer of 20 nm gold [21] . SEM-samples were examined in a Philips SEM 505 at 30 kV.…”

Section: Liver Tissue Preparation For Light and Electron Microscopymentioning

confidence: 99%

Influence of kavain on hepatic ultrastructure

Fu¹,

Korkmaz²,

Braet³

et al. 2008

WJG

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AIM:To investigate whether the major kavalactone k a v a i n i m p o s e s a d v e r s e e f f e c t s o n t h e l i v e r ultrastructure and function by affecting vascular and microvascular architecture and altering hepatocellular morphology. METHODS:Kavain solution (10 µg/mL or 43.5 µmol/L) was perfused for 2 h in isolated rat livers. After standard fixation and tissue preparation, the samples were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and light microscopy (LM).RESULTS: LM, SEM, and TEM examinations indicated kavain-treated rat livers (n = 4) displayed severe vascular and endothelial damage compared to control livers (n = 4). CONCLUSION:The data so far support the hypothesis that kavain induces adverse effects on liver; additional investigations with other kavalactones and their effects on liver are urgently needed.

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Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Cited by 287 publications

References 8 publications

Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

Vascular growth factor binding kinetics to the endothelial cell basement membrane, with a kinetics-based correction for substrate binding

Influence of kavain on hepatic ultrastructure

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