Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical‐point drying methods for scanning electron microscopy of biological specimens

Bray, D. F.; Bagu, John R.; Koegler, P.

doi:10.1002/jemt.1070260603

Cited by 246 publications

(166 citation statements)

References 9 publications

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“…To rule out that the structures were an artifact caused by the dehydration process, we analyzed kidney samples dehydrated with hexamethyldisilazane (HMDS) to reach critical point dehydration less aggressively. 22,23 Renal specimens treated with HMDS ( Figure 1F) showed the same heteroporous structure as samples dehydrated by a critical point dryer ( Figure 1, D and E). In addition, to exclude the possibility that gold-coating could induce artifacts in the ultrastructure of the filtration slit, we also imaged uncoated samples of the peripheral glomerular capillary wall.…”

Section: The In-lens Detector Strengthens Sem Imagingmentioning

confidence: 66%

“…Immediately after dehydration, samples were rinsed with liquid CO 2 with a Bal-Tec 030 critical point dryer (BAL-TEC AG, Balzers, Liechtenstein) or dehydrated in pure HMDS (Polyscience) (twice for 0.5 hours), which permitted reaching the critical point of dehydration in a less aggressive way. 22,23 Samples were mounted on stubs and coated with gold in a sputter coater (Agar Scientific, Stansted, UK). In parallel, some kidney samples were used for SEM without gold coating, for comparison.…”

Section: Semmentioning

confidence: 99%

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Imaging of the Porous Ultrastructure of the Glomerular Epithelial Filtration Slit

Gagliardini

Conti

Benigni

et al. 2010

Journal of the American Society of Nephrology

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The ultrastructure of the glomerular filtration slit is still controversial. In the last 30 years, observations from transmission electron microscopy (TEM) and theoretical analysis of solute clearance produced conflicting results. Here, we used scanning EM with a high-sensitivity detector to image the deepest regions of the filtration slits and report a previously undescribed organization of the slits' ultrastructure. In contrast to previous TEM imaging, we observed circular and ellipsoidal pores in the podocyte junctions mainly located in the central region of the slit diaphragm. The normal mean pore radius estimated by digital morphometric analysis had a log-normal distribution, with an average value of 12.1 nm. In proteinuric pathologic conditions, the mean pore radius values were also log-normally distributed with the presence of some very large pores, exceeding the sizes observed in normal conditions. Our morphologic analysis suggests that the filtration slit is a heteroporous structure instead of the previously proposed zipper-like structure. Selective changes in the ultrastructural organization of the pores may be responsible for the increased filtration of plasma proteins in glomerular disease. The permeability properties of the glomerular capillary wall allow the filtration of high water flow and small solutes but efficient retention of plasma proteins the size of albumin or larger. 1 It has been indicated that most of the restriction of macromolecule ultrafiltration is caused by the epithelial filtration slits, because plasma proteins can cross endothelial cells and glomerular basement membrane (GBM) more easily. 2 Changes in glomerular permselective function resulting in increased glomerular filtration of plasma proteins and incomplete tubular reabsorption are responsible for loss of proteins in the urine. These events have been linked to the progression of renal diseases. 3 With the aim to characterize the mechanisms behind proteinuria, in the last 30 years, solute clearance techniques have been used to estimate the selective properties of the glomerular barrier in experimental models of nephropathy and in patients. In particular, glomerular size-selective function has been studied using renal clearance of neutral test macromolecules (i.e., neutral dextran and Ficoll) that are not secreted or reabsorbed at the tubular level. Theoretical models of glomerular size selectivity have been conventionally used to derive intrinsic membrane selective properties from fractional clearance of test macromolecules in terms of density and size of hypothetical pores perforating the glomerular capillary wall. 2,4,5 Thus, according to the two-pore

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Section: The In-lens Detector Strengthens Sem Imagingmentioning

confidence: 66%

Section: Semmentioning

confidence: 99%

Imaging of the Porous Ultrastructure of the Glomerular Epithelial Filtration Slit

Gagliardini

Conti

Benigni

et al. 2010

Journal of the American Society of Nephrology

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“…4 of Lee et al [12]). However, since freeze-drying is known to disrupt soft structures [18], we employ a gentler method of preparation: incremental solvent exchange from water to ethanol to HMDS, which is known [19] to result in equal structural preservation as critical point drying.…”

Section: Imaging Of Natural Plaquesmentioning

confidence: 99%

The microscopic network structure of mussel ( Mytilus ) adhesive plaques

Filippidi

DeMartini

Molina

et al. 2015

J. R. Soc. Interface.

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Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 mm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.

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“…without Triton). The sample was then dehydrated by successive few minutes dipping in 50% (diluted with water) then 100% acetone followed by immersion in 50% (diluted with acetone) then 100% hexamethyldisilazane (HMDS) 14 . Samples were finally allowed to dry slowly under a hood.…”

Section: -Neuronal Culture and Labelingmentioning

confidence: 99%

Nanoscale Surface Topography Reshapes Neuronal Growth in Culture

et al. 2014

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Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nano-pillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nano-pillars with a typical distance between adhesion points of about 500nm. These neurons produce less neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted to a differential surface topography, neurons specify an axon preferentially onto nano-pillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.

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Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical‐point drying methods for scanning electron microscopy of biological specimens

Cited by 246 publications

References 9 publications

Imaging of the Porous Ultrastructure of the Glomerular Epithelial Filtration Slit

Imaging of the Porous Ultrastructure of the Glomerular Epithelial Filtration Slit

The microscopic network structure of mussel ( Mytilus ) adhesive plaques

Nanoscale Surface Topography Reshapes Neuronal Growth in Culture

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