Artifacts caused by dehydration and epoxy embedding in TEM

1991 ◽  
Vol 49 ◽  
pp. 338-339
Author(s):  
Hilton H. Mollenhauer
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Volume Change ◽  
Tissue Damage ◽  
Beam Specimen ◽  
Chemical Fixation ◽  
Resin Impregnation ◽  
Storage Vesicles ◽  
A Cell ◽  
Tissue Shrinkage

Various means have been devised to preserve biological specimens for electron microscopy, the most common being chemical fixation followed by dehydration and resin impregnation. It is intuitive, and has been amply demonstrated, that these manipulations lead to aberrations of many tissue elements. This report deals with three parts of this problem: specimen dehydration, epoxy embedding resins, and electron beam-specimen interactions. However, because of limited space, only a few points can be summarized.Dehydration: Tissue damage, or at least some molecular transitions within the tissue, must occur during passage of a cell or tissue to a nonaqueous state. Most obvious, perhaps, is a loss of lipid, both that which is in the form of storage vesicles and that associated with tissue elements, particularly membranes. Loss of water during dehydration may also lead to tissue shrinkage of 5-70% (volume change) depending on the tissue and dehydrating agent.

scholarly journals Fluorescent and Electron-Dense Green Color Emitting Nanodiamonds for Single-Cell Correlative Microscopy

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5897 ◽  
Author(s):  
Neeraj Prabhakar ◽  
Markus Peurla ◽  
Olga Shenderova ◽  
Jessica M. Rosenholm
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Fluorescent Properties ◽  
Green Color ◽  
Chemical Treatments ◽  
A Cell ◽  
Green Fluorescent ◽  
Microscopy Images

Correlative light and electron microscopy (CLEM) is revolutionizing how cell samples are studied. CLEM provides a combination of the molecular and ultrastructural information about a cell. For the execution of CLEM experiments, multimodal fiducial landmarks are applied to precisely overlay light and electron microscopy images. Currently applied fiducials such as quantum dots and organic dye-labeled nanoparticles can be irreversibly quenched by electron beam exposure during electron microscopy. Generally, the sample is therefore investigated with a light microscope first and later with an electron microscope. A versatile fiducial landmark should offer to switch back from electron microscopy to light microscopy while preserving its fluorescent properties. Here, we evaluated green fluorescent and electron dense nanodiamonds for the execution of CLEM experiments and precisely correlated light microscopy and electron microscopy images. We demonstrated that green color emitting fluorescent nanodiamonds withstand electron beam exposure, harsh chemical treatments, heavy metal straining, and, importantly, their fluorescent properties remained intact for light microscopy.

Techniques for Transmission Electron Microscopy of Fiber-Matrix Interfaces in Aluminum Alloy-Boron Composites by Ion Erosio

1971 ◽  
Vol 29 ◽  
pp. 204-205
Author(s):  
G. G. Shaw
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Aluminum Alloy ◽  
Electron Beam ◽  
Pure Aluminum ◽  
Ion Milling ◽  
Transmission Electron ◽  
Fiber Matrix ◽  
Ion Erosion ◽  
Row Of Fibers

The morphology and composition of the fiber-matrix interface can best be studied by transmission electron microscopy and electron diffraction. For some composites satisfactory samples can be prepared by electropolishing. For others such as aluminum alloy-boron composites ion erosion is necessary.When one wishes to examine a specimen with the electron beam perpendicular to the fiber, preparation is as follows: A 1/8 in. disk is cut from the sample with a cylindrical tool by spark machining. Thin slices, 5 mils thick, containing one row of fibers, are then, spark-machined from the disk. After spark machining, the slice is carefully polished with diamond paste until the row of fibers is exposed on each side, as shown in Figure 1.In the case where examination is desired with the electron beam parallel to the fiber, preparation is as follows: Experimental composites are usually 50 mils or less in thickness so an auxiliary holder is necessary during ion milling and for easy transfer to the electron microscope. This holder is pure aluminum sheet, 3 mils thick.

Epoxy Resin Embedment

1968 ◽  
Vol 26 ◽  
pp. 100-101
Author(s):  
J. G. Adams ◽  
M. M. Campbell ◽  
H. Thomas ◽  
J. J. Ghldonl
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Epoxy Resin ◽  
Light Microscope ◽  
Epoxy Resins ◽  
Image Contrast ◽  
Tissue Preservation ◽  
Resin System ◽  
Excellent Quality

Since the introduction of epoxy resins as embedding material for electron microscopy, the list of new formulations and variations of widely accepted mixtures has grown rapidly. Described here is a resin system utilizing Maraglas 655, Dow D.E.R. 732, DDSA, and BDMA, which is a variation of the mixtures of Lockwood and Erlandson. In the development of the mixture, the Maraglas and the Dow resins were tested in 3 different volumetric proportions, 6:4, 7:3, and 8:2. Cutting qualities and characteristics of stability in the electron beam and image contrast were evaluated for these epoxy mixtures with anhydride (DDSA) to epoxy ratios of 0.4, 0.55, and 0.7. Each mixture was polymerized overnight at 60°C with 2% and 3% BDMA.Although the differences among the test resins were slight in terms of cutting ease, general tissue preservation, and stability in the beam, the 7:3 Maraglas to D.E.R. 732 ratio at an anhydride to epoxy ratio of 0.55 polymerized with 3% BDMA proved to be most consistent. The resulting plastic is relatively hard and somewhat brittle which necessitates trimming and facing the block slowly and cautiously to avoid chipping. Sections up to about 2 microns in thickness can be cut and stained with any of several light microscope stains and excellent quality light photomicrographs can be taken of such sections (Fig. 1).

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

Aberrant Type C Virus Production in a Cell Line Derived from Balb/3T3

1972 ◽  
Vol 30 ◽  
pp. 286-287
Author(s):  
N. Savage ◽  
A. Hackett
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Leukemia Virus ◽  
Morphological Characteristics ◽  
Virus Production ◽  
Oncogenic Viruses ◽  
Endogenous Virus ◽  
Virus Particles ◽  
Moloney Leukemia Virus ◽  
A Cell

A cell line, UC1-B, which was derived from Balb/3T3 cells, maintains the same morphological characteristics of the non-transformed parental culture, and shows no evidence of spontaneous virus production. Survey by electron microscopy shows that the cell line consists of spindle-shaped cells with no unusual features and no endogenous virus particles.UC1-B cells respond to Moloney leukemia virus (MLV) infection by a change in morphology and growth pattern which is typical of cells transformed by sarcoma virus. Electron microscopy shows that the cells are now variable in shape (rounded, rhomboid, and spindle), and each cell type has some microvilli. Virtually all (90%) of the cells show virus particles developing at the cell surface and within the cytoplasm. Maturing viruses, typical of the oncogenic viruses, are found along with atypical tubular forms in the same cell.

Head Size and DNA Content in Bacteriophage T4

1972 ◽  
Vol 30 ◽  
pp. 200-201
Author(s):  
Fred Eiserling ◽  
A. H. Doermann ◽  
Linde Boehner
Keyword(s):  
Electron Microscopy ◽  
Dna Content ◽  
Bacteriophage T4 ◽  
Head Size ◽  
Virus Particles ◽  
Altered Protein ◽  
A Cell ◽  
Bacterial Viruses ◽  
A Site ◽  
Protein Shell

The control of form or shape inheritance can be approached by studying the morphogenesis of bacterial viruses. Shape variants of bacteriophage T4 with altered protein shell (capsid) size and nucleic acid (DNA) content have been found by electron microscopy, and a mutant (E920g in gene 66) controlling head size has been described. This mutant produces short-headed particles which contain 2/3 the normal DNA content and which are non-viable when only one particle infects a cell (Fig. 1).We report here the isolation of a new mutant (191c) which also appears to be in gene 66 but at a site distinct from E920g. The most striking phenotype of the mutant is the production of about 10% of the phage yield as “giant” virus particles, from 3 to 8 times longer than normal phage (Fig. 2).

New type electron gun with electrostatic and electromagnetic lenses

1978 ◽  
Vol 36 (1) ◽  
pp. 62-63
Author(s):  
T. Ichinokawa ◽  
H. Maeda
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Optimum Condition ◽  
Bias Voltage ◽  
Electron Gun ◽  
Charge Effect ◽  
Micro Fabrication ◽  
Maximum Brightness ◽  
New Type ◽  
The Ideal

I. IntroductionThermionic electron gun with the Wehnelt grid is popularly used in the electron microscopy and electron beam micro-fabrication. It is well known that this gun could get the ideal brightness caluculated from the Lengumier and Richardson equations under the optimum condition. However, the design and ajustment to the optimum condition is not so easy. The gun has following properties with respect to the Wehnelt bias; (1) The maximum brightness is got only in the optimum bias. (2) In the larger bias than the optimum, the brightness decreases with increasing the bias voltage on account of the space charge effect. (3) In the smaller bias than the optimum, the brightness decreases with bias voltage on account of spreading of the cross over spot due to the aberrations of the electrostatic immersion lens.In the present experiment, a new type electron gun with the electrostatic and electromagnetic lens is designed, and its properties are examined experimentally.

Effects of High-Energy Ions on Synthetic Quartz

1972 ◽  
Vol 30 ◽  
pp. 544-545
Author(s):  
Joseph J. Comer ◽  
Charles Bergeron ◽  
Lester F. Lowe
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Beam ◽  
High Energy ◽  
Transmission Electron ◽  
Kikuchi Lines ◽  
High Energy Ions ◽  
Diffraction Patterns ◽  
Dauphiné Twinning ◽  
Beam Damage

Using a Van De Graaff Accelerator thinned specimens were subjected to bombardment by 3 MeV N+ ions to fluences ranging from 4x1013 to 2x1016 ions/cm2. They were then examined by transmission electron microscopy and reflection electron diffraction using a 100 KV electron beam.At the lowest fluence of 4x1013 ions/cm2 diffraction patterns of the specimens contained Kikuchi lines which appeared somewhat broader and more diffuse than those obtained on unirradiated material. No damage could be detected by transmission electron microscopy in unannealed specimens. However, Dauphiné twinning was particularly pronounced after heating to 665°C for one hour and cooling to room temperature. The twins, seen in Fig. 1, were often less than .25 μm in size, smaller than those formed in unirradiated material and present in greater number. The results are in agreement with earlier observations on the effect of electron beam damage on Dauphiné twinning.

High Resolution Scanning Electron Microscopy

1991 ◽  
Vol 49 ◽  
pp. 478-479
Author(s):  
David Joy ◽  
James Pawley
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Beam ◽  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Electron Probe ◽  
Impact Point ◽  
Interaction Volume ◽  
Scanning Electron

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).

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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