Preparation of biological monolayers for producing high-resolution scanning electron micrographs Code: PLOS2021 [PONE-D-21-26669] - [EMID:f8569bc7b537f6e4] v1

Author(s):  
Shireen Mentor ◽  
Franscious Cummings ◽  
David Fisher ◽  
shireen.mentor not provided

Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, viz., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

Author(s):  
D. Johnson ◽  
P. Moriearty

Since several species of Schistosoma, or blood fluke, parasitize man, these trematodes have been subjected to extensive study. Light microscopy and conventional electron microscopy have yielded much information about the morphology of the various stages; however, scanning electron microscopy has been little utilized for this purpose. As the figures demonstrate, scanning microscopy is particularly helpful in studying at high resolution characteristics of surface structure, which are important in determining host-parasite relationships.


Author(s):  
David Joy ◽  
James Pawley

The scanning electron microscope (SEM) builds up an image by sampling contiguous sub-volumes near the surface of the specimen. A fine electron beam selectively excites each sub-volume and then the intensity of some resulting signal is measured. The spatial resolution of images made using such a process is limited by at least three factors. Two of these determine the size of the interaction volume: the size of the electron probe and the extent to which detectable signal is excited from locations remote from the beam impact point. A third limitation emerges from the fact that the probing beam is composed of a finite number of discrete particles and therefore that the accuracy with which any detectable signal can be measured is limited by Poisson statistics applied to this number (or to the number of events actually detected if this is smaller).


Author(s):  
Yun Lu ◽  
David C. Joy

High resolution scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) were performed to investigate microparticles in blended cements and their hydration products containing sodium-rich chemical wastes. The physical appearance of powder particles and the morphological development at different hydration stages were characterized by using high resolution SEM Hitachi S-900 and by SEM S-800 with a EDX spectrometer. Microparticles were dispersed on the sample holder and glued by 1% palomino solution. Hydrated bulk samples were dehydrated by acetone and mounted on the holder by silver paste. Both fracture surfaces and flat cutting sections of hydrating samples were prepared and examined. Some specimens were coated with an 3 nm thick Au-Pd or Cr layer to provide good conducting surfaces. For high resolution SEM S-900 observations the accelerating voltage of electrons was 1-2 KeV to protect the electron charging. Microchemical analyses were carried out by S800/EDS equipped with a LINK detector of take-off angle =40°.


Author(s):  
William J. Lamoreaux ◽  
David L. Smalley ◽  
Larry M. Baddour ◽  
Alfred P. Kraus

Infections associated with the use of intravascular devices have been documented and have been reported to be related to duration of catheter usage. Recently, Eaton et al. reported that Staphylococcus epidermidis may attach to silastic catheters used in continuous ambulatory peritoneal dialysis (CAPD) treatment. The following study presents findings using scanning electron microscopy (SEM) of S. epidermidis adherence to silastic catheters in an in vitro model. In addition, sections of polyvinyl chloride (PVC) dialysis bags were also evaluated by SEM.The S. epidermidis strain RP62A which had been obtained in a previous outbreak of coagulase-negative staphylococcal sepsis at local hospitals was used in these experiments. The strain produced surface slime on exposure to glucose, whereas a nonadherent variant RP62A-NA, which was also used in these studies, failed to produce slime. Strains were grown overnight on blood agar plates at 37°C, harvested from the surface and resuspended in sterile saline (0.85%), centrifuged (3,000 rpm for 10 minutes) and then washed twice in 0.1 M phosphate-buffered saline at pH 7.0. Organisms were resuspended at a concentration of ca. 106 CFU/ml in: a) sterile unused dianeal at 4.25% dextrose, b) sterile unused dianeal at 1.5% dextrose, c) sterile used dialysate previously containing 4.25% dextrose taken from a CAPD patient, and d) sterile used dialysate previously containing 1.5% dextrose taken from a CAPD patient.


Author(s):  
Ya Chen ◽  
Geoffrey Letchworth ◽  
John White

Low-temperature high-resolution scanning electron microscopy (cryo-HRSEM) has been successfully utilized to image biological macromolecular complexes at nanometer resolution. Recently, imaging of individual viral particles such as reovirus using cryo-HRSEM or simian virus (SIV) using HRSEM, HV-STEM and AFM have been reported. Although conventional electron microscopy (e.g., negative staining, replica, embedding and section), or cryo-TEM technique are widely used in studying of the architectures of viral particles, scanning electron microscopy presents two major advantages. First, secondary electron signal of SEM represents mostly surface topographic features. The topographic details of a biological assembly can be viewed directly and will not be obscured by signals from the opposite surface or from internal structures. Second, SEM may produce high contrast and signal-to-noise ratio images. As a result of this important feature, it is capable of visualizing not only individual virus particles, but also asymmetric or flexible structures. The 2-3 nm resolution obtained using high resolution cryo-SEM made it possible to provide useful surface structural information of macromolecule complexes within cells and tissues. In this study, cryo-HRSEM is utilized to visualize the distribution of glycoproteins of a herpesvirus.


Author(s):  
W.W. Adams ◽  
G. Price ◽  
A. Krause

It has been shown that there are numerous advantages in imaging both coated and uncoated polymers in scanning electron microscopy (SEM) at low voltages (LV) from 0.5 to 2.0 keV compared to imaging at conventional voltages of 10 to 20 keV. The disadvantages of LVSEM of degraded resolution and decreased beam current have been overcome with the new generation of field emission gun SEMs. In imaging metal coated polymers in LVSEM beam damage is reduced, contrast is improved, and charging from irregularly shaped features (which may be unevenly coated) is reduced or eliminated. Imaging uncoated polymers in LVSEM allows direct observation of the surface with little or no charging and with no alterations of surface features from the metal coating process required for higher voltage imaging. This is particularly important for high resolution (HR) studies of polymers where it is desired to image features 1 to 10 nm in size. Metal sputter coating techniques produce a 10 - 20 nm film that has its own texture which can obscure topographical features of the original polymer surface. In examining thin, uncoated insulating samples on a conducting substrate at low voltages the effect of sample-beam interactions on image formation and resolution will differ significantly from the effect at higher accelerating voltages. We discuss here sample-beam interactions in single crystals on conducting substrates at low voltages and also present the first results on HRSEM of single crystal morphologies which show some of these effects.


1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.


2018 ◽  
Author(s):  
Grigore Moldovan ◽  
Wolfgang Joachimi ◽  
Guillaume Boetsch ◽  
Jörg Jatzkowski ◽  
Frank Altman

Abstract This work presents advanced resistance mapping techniques based on Scanning Electron Microscopy (SEM) with nanoprobing systems and the related embedded electronics. Focus is placed on recent advances to reduce noise and increase speed, such as integration of dedicated in situ electronics into the nanoprobing platform, as well as an important transition from current-sensitive to voltagesensitive amplification. We show that it is now possible to record resistance maps with a resistance sensitivity in the 10W range, even when the total resistance of the mapped structures is in the range of 100W. A reference structure is used to illustrate the improved performance, and a lowresistance failure case is presented as an example of analysis made possible by these developments.


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