Field Emission Scanning Electron Microscopy Of Mouse Cerebellar Synaptic Contacts

Samples of albino mice cerebellar cortex were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and inmersed in 4% glutaraldehyde in O. l M phosphate buffer solution, pH 7.4, for 24h at 4°C; and postfixed for 1 h in a similarly buffered 1% osmium tetroxide solution. Specimens were dehydrated in a graded serie of ethanol (30, 50, 70, 80, 90 2x100%) prior to wrapping individual tissue pieces in preformed absolute ethanol filled parafilm cryofracture packets. Rapid freezing of packets was performed by plunging into LN2. First, the packet was transferred from the LN2 storage vessel with LNT chilled forceps in order to avoid themial damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool.

The Effect of Incomplete Fixation of Elastin on the Appearance of Pulmonary Alveoli

Pulmonary alveolar geometry in histological sections prepared by quick-freezing the lung, with freeze-substitution of ice, fixation of the dry lung with osmium tetroxide in nonaqueous and nonpolar solvent and celloidin embedding is remarkably different from the geometry seen in preparations made by instilling aqueous aldehyde or osmium tetroxide solution in the airways. The alveolar walls in the quick-frozen lung are linear; those in the others are wrinkled. The difference was demonstrated to be due to incomplete fixation of elastin.

Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.

Ultrahistochemical Studies of Mucosubstances : Electron Microscope Observation and Microprobe Analysis of the Chick Embryo Cornea

As described in a previous report, Alcian blue provides the selective contrast enhancement of interfibrillar substances of the mesostroma of the chick embryo cornea. This finding was also verified by the electron probe microanalysis of adjacent sections. In the present study the nature of interfibrillar substances was ultrahistochemically examined by a modified Seligman's method for the demonstration of some oxidizable glycols.Strips of the 4th day chick embryo.cornea were fixed in ice-cold 2.5% glutaraldehyde solution (pH 7.4). Tissue specimens treated with 1% aqueous periodic acid or 0.2% hyaluronidase solution of pH 6.0 for 15 min were transferred into 1% thiocarbohydrazide, followed by a rinse in 1% osmium tetroxide solution (60°C, 50 min).

ELEMENTAL ANALYSIS OF PRECIPITATES FORMED IN NUCLEI BY ANTIMONATE-OSMIUM TETROXIDE FIXATION

Elements retained in cervical lymph nodes, isolated hepatic nuclei and salt-impregnated gels by fixation with antimonate- or pyrophosphate-containing and other osmium tetroxide solutions were assayed by nuclear activation analysis or by atomic absorption spectrophotometry. Salts preserved by the antimonate-osmium tetroxide fixative in lymph nodes, isolated nuclei and a KCl-enriched gel consisted almost entirely of potassium antimonate. The K+ in the precipitates in these specimens appeared to derive partially from that in the fixative solution and partially from that in the specimen. Salts preserved by the antimonate-osmium tetroxide fixative in an NaCl-supplemented gel consisted partly of potassium antimonate derived from the fixative as in unsupplemented gels and partly of sodium antimonate. The Na+ precipitated in this gel amounted to less than one-half that originally present. In comparison the pyrophosphate-osmium tetroxide solution retained higher levels of K+ in lymph nodes, nuclei and the KCl gel, but the potassium pyrophosphate was not evident as electron-opaque precipitates. The latter fixative was less effective in preserving Na+ in the NaCl gel. The pyrophosphate-containing fixative, which was about twice as efficient as the antimonate-containing solution in retaining the divalent cations, preserved 70% of the Mg++ and 100% of the Ca++ added to gels.

Biological Applications of the Scanning Transmitted Electron Microscope

Recently, very thick section of biological specimens have been observed by using super high voltage electron microscope to obtain a three dimensional model of a specimen.However, the thicker the section, the more difficult the penetration of staining solution into the depth of thick section. So, it is doutful whether the stain solution fully penetrated into the thick section. This paper reports an observation of various thick unstained sections using scanning transmitted electron microscope(STEM) and conventional electron microscope(CEM).The observed specimen is mice liver cells. The tissue was fixed in cold 1% phosphate-buffered osmium tetroxide solution for 2 hours. After fixation, the tissue was dehydrated in a graded series of ethanols and then embedded in Epon 812. The 500 Å, 5,000 Å and 1μ thick sections were cut, not stained with uranyle and lead, and then examined with STEM and GEM operated at accelerating voltage 80 kV.

AN ELECTRON MICROSCOPIC CHARACTERIZATION OF CLASSES OF SYNAPTIC VESICLES BY MEANS OF CONTROLLED ALDEHYDE FIXATION

Examination of variables of aldehyde fixation that may affect the shape of agranular synaptic vesicles has revealed that even brief storage of aldehyde-perfused nervous tissue pieces in cacodylate buffer, prior to hardening in osmium tetroxide, has an unusually severe flattening effect on agranular vesicles of a particular type. These are the vesicles of peripheral cholinergic axon endings, and of certain central synaptic bulbs. Types of synaptic bulbs can now be further defined on the basis of shape of agranular synaptic vesicles under controlled conditions of aldehyde fixation. Previously described "S" bulbs in the spinal cord contain uniformly spheroid vesicles, which are wholly resistant to flattening. Previously described "F" bulbs contain somewhat smaller agranular vesicles that are flattened after aldehyde fixation, even when this is followed by prompt hardening in osmium tetroxide solution. A third type, previously characterized as having irregularly round agranular vesicles after the above treatment, contains only severely flattened vesicles when the osmium tetroxide hardening is preceded by even a brief wash with sodium cacodylate buffer containing sucrose. Moreover, the "third" type is characteristic of all cholinergic peripheral axon endings examined, as well as the large axosomatic ("L") synaptic bulbs of the spinal cord.

ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCE AND ANTIMONATE-PRECIPITABLE CATION IN HUMAN AND RABBIT PLATELETS AND MEGAKARYOCYTES

For selective ultrastructural localization of acid mucosubstance in rabbit and human platelets and megakaryocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde or osmium tetroxide, sectioned at 40 µ and stained with the Rinehart-Abul-Haj solution of dialyzed iron. In specimens from both rabbit and man, dialyzed iron staining was observed within nucleoids of the cytoplasmic granules (α-granulomeres) of platelets and megakaryocytes, on the outer surface of the plasma membranes of platelets and megakaryocytes and on the luminal surface of demarcation membranes of megakaryocytes. These results were obtained following any of the three fixation procedures, except when nucleoids failed to stain after glutaraldehyde fixation. For ultrastructural localization of pyroantimonate-precipitable cation, bone marrow and buffy coat specimens were fixed in Komnick's solution of potassium pyroantimonate and osmium tetroxide. In specimens from both species, antimonate deposits were localized within the dense bodies (5-hydroxytryptamine organelles) of platelets and within nucleoids of cytoplasmic granules of platelets and megakaryocytes. The dense bodies were well preserved in platelets fixed in a pyrophosphate-osmium tetroxide solution but were poorly, if at all, preserved by osmium tetroxide solutions containing other buffers.

ELECTRON PROBE ANALYSIS OF CATIONIC SPECIES IN PYROANTIMONATE PRECIPITATES IN EPON-EMBEDDED TISSUE

Electron microscopy of Epon-embedded mouse vas deferens eipthelium treated with buffered potassium pyroantimonate-osmium tetroxide solution revealed precipitates in the lamina propria and along the apical plasma membrane. Electron microprobe elemental analysis of adjacent sections demonstrated that the deposits contained sodium and antimony. Other cations noted to precipitate pyroantimonate in vitro were not present in large amounts compared to controls, and were randomly distributed.