scholarly journals MEASUREMENT OF THICKNESS WITHIN SECTIONS BY QUANTITATIVE ELECTRON MICROSCOPY

1969 ◽  
Vol 40 (3) ◽  
pp. 768-772 ◽  
Author(s):  
Lloyd Silverman ◽  
Berit Schreiner ◽  
David Glick
Keyword(s):  
Electron Microscopy ◽  
Phosphotungstic Acid ◽  
Exchange Resin ◽  
Interference Microscopy ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Calculated Mass ◽  
Thickness Standard ◽  
Embedding Medium ◽  
Measurement Of Thickness

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.

scholarly journals MEASUREMENT OF PROTEIN CONCENTRATION BY QUANTITATIVE ELECTRON MICROSCOPY

1969 ◽  
Vol 40 (3) ◽  
pp. 773-778 ◽  
Author(s):  
Lloyd Silverman ◽  
David Glick
Keyword(s):  
Electron Microscopy ◽  
Protein Concentration ◽  
Clinical Laboratory ◽  
Red Cells ◽  
Cell Protein ◽  
Red Cell ◽  
Protein Loss ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Mean Values

The method of quantitative electron microscopy was applied to the measurement of protein concentration in thin sections. The human erythrocyte was selected as a model because of its apparently uniform protein concentration. Phosphotungstic acid (PTA) in aqueous solution was used as a reversible stain for protein, and PTA-stained Dowex resin spheres were embedded along with the red cells as standards for measurement of section thickness. The mass of stain removed from a given area of sectioned red cell by buffer (pH 7.4) was measured by quantitative electron microscopy. From the stoichiometry of the reaction between PTA and red cell protein established in this study, the amount of protein present in the measured area was calculated. From this amount of protein and the measured thickness, the concentration of protein was calculated and expressed as g/100 ml, for comparison with the clinical laboratory value for hemoglobin. Groups of red cells from the same sample were measured on 3 different days and their mean values (g/100 ml ± SD) were 29 ± 3.9, 30 ± 2.7, and 33 ± 4.6, compared to the clinical laboratory value of 32.1 g/100 ml packed cells, after correction for volume change and protein loss during fixation.

scholarly journals An Effective Method of Preparing Sections of Bacillus polymyxa Sporangia and Spores for Electron Microscopy

1960 ◽  
Vol 7 (2) ◽  
pp. 373-376 ◽  
Author(s):  
Pauline E. Holbert
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Micrographs ◽  
Epoxy Resins ◽  
Bacillus Polymyxa ◽  
Thin Sections ◽  
Similar Images ◽  
Embedding Medium ◽  
First Time ◽  
Diamond Knife

Bacillus polymyxa sporangia and spores were prepared for examination in the electron microscope by methods whose critical features were apparently: judicious use of vacuum, to encourage complete penetration of the embedding medium; the use of epoxy resins as embedding media; and cutting of the thin sections with a diamond knife. Electron micrographs of material prepared in this manner exhibit undeformed sporangial sections. Some of the structures revealed have been shown before, though perhaps less distinctly; other structures are revealed here for the first time. While this single study does not pretend to elucidate all the complexities of sporulation in bacteria, these and similar images should make this possible, and some mention of the preparatory techniques that lead to them seems advisable at this time.

scholarly journals ELECTRON MICROSCOPE AND EXPERIMENTAL INVESTIGATIONS OF THE NEUROFILAMENTOUS NETWORK IN DEITERS' NEURONS

1974 ◽  
Vol 61 (3) ◽  
pp. 701-722 ◽  
Author(s):  
J. Metuzals ◽  
W. E. Mushynski
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Interference Microscopy ◽  
Experimental Investigations ◽  
Rabbit Brain ◽  
Nuclear Pores ◽  
Isolated Cells ◽  
Thin Sections ◽  
Space Network ◽  
Neurofilamentous Network

The assembly of filamentous elements and their relations to the plasma membrane and to the nuclear pores have been studied in Deiters' neurons of rabbit brain. Electron microscopy of thin sections and of ectoplasm spread preparations have been integrated with physicochemical experiments and differential interference microscopy of freshly isolated cells. A neurofilamentous network extends as a continuous, three-dimensional, semilattice structure throughout the ectoplasm, the "plasma roads," and the perinuclear zone of the perikaryon. This space network consists of ∼90-Å wide neurofilaments arranged in fascicles which are interconnected by an exchange of neurofilaments. The neurofilaments consist of intercoiled ∼20-Å wide unit-filaments and are associated through cross-associating filaments with other neurofilaments of the fascicle and with microfilaments. The ∼20–50-Å wide microfilaments display intimate associations with the plasma membrane and with the nuclear pores. Electron microscopy of thin sections from glycerinated and heavy meromyosin-treated Deiters' neurons shows that actin-like filaments are present in the pre- and postsynaptic regions of synapses terminating on these neurons. It is proposed that the neurofilamentous space network serves a transducing function by linking plasma membrane activities with the genetic machinery of the neuron.

scholarly journals THE REACTIVITY AND STAINING OF TISSUE PROTEINS WITH PHOSPHOTUNGSTIC ACID

1969 ◽  
Vol 40 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Lloyd Silverman ◽  
David Glick
Keyword(s):  
Phosphotungstic Acid ◽  
Negative Contrast ◽  
Mitochondrial Matrix ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Minimal Distortion ◽  
Fixation Treatment ◽  
Intense Electron ◽  
Positively Charged ◽  
Aqueous Acidic Medium

After aldehyde-fixation, treatment with phosphotungstic acid (PTA) in aqueous acidic medium was shown to produce an intense electron-opaque stain with minimal distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic reticulum, and the Z-band of muscle were densely stained, whereas membranes stood out in negative contrast. Staining of glycogen or lipid was not apparent. Under certain conditions the stain density reflected the concentration of protein based on the quantitative reaction of PTA with the positively charged groups, although the stoichiometry of the reaction between PTA and protein varied with the kind of protein. The staining conditions established should provide a base for the use of the method in quantitative electron microscopy, particularly on thin sections.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

Ectodesmata as Revealed By Freeze-Etch Preparations of Plant Epidermal Cell Walls

1973 ◽  
Vol 31 ◽  
pp. 468-469
Author(s):  
N.C. Lyon ◽  
W. C. Mueller
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Nitric Acid ◽  
Oxalic Acid ◽  
Light Microscopy ◽  
Epidermal Cell ◽  
Cell Walls ◽  
Plant Viruses ◽  
Plant Leaves ◽  
Thin Sections

Schumacher and Halbsguth first demonstrated ectodesmata as pores or channels in the epidermal cell walls in haustoria of Cuscuta odorata L. by light microscopy in tissues fixed in a sublimate fixative (30% ethyl alcohol, 30 ml:glacial acetic acid, 10 ml: 65% nitric acid, 1 ml: 40% formaldehyde, 5 ml: oxalic acid, 2 g: mecuric chloride to saturation 2-3 g). Other workers have published electron micrographs of structures transversing the outer epidermal cell in thin sections of plant leaves that have been interpreted as ectodesmata. Such structures are evident following treatment with Hg++ or Ag+ salts and are only rarely observed by electron microscopy. If ectodesmata exist without such treatment, and are not artefacts, they would afford natural pathways of entry for applied foliar solutions and plant viruses.

Albumins as Embedding Media for Electron Microscopy

1969 ◽  
Vol 27 ◽  
pp. 422-423 ◽  
Author(s):  
J. L. Farrant ◽  
J. D. McLean
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Temperature ◽  
Biological Material ◽  
Globular Proteins ◽  
The Past ◽  
Antibody Staining ◽  
Thin Sectioning ◽  
Embedding Medium

For electron microscope techniques such as ferritin-labeled antibody staining it would be advantageous to have available a simple means of thin sectioning biological material without subjecting it to lipid solvents, impregnation with plastic monomers and their subsequent polymerization. With this aim in view we have re-examined the use of protein as an embedding medium. Gelatin which has been used in the past is not very satisfactory both because of its fibrous nature and the high temperature necessary to keep its solutions fluid. We have found that globular proteins such as the serum and egg albumins can be cross-linked so as to yield blocks which are suitable for ultrathin sectioning.

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