Microscopic Analysis of Oxidative Stress in Cultured Cells. II. Transmission Electron Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 636-637
Author(s):  
Tracy L. Gales ◽  
Beverly E. Maleeff ◽  
Padma K. Narayanan ◽  
Mark A. Tirmenstein ◽  
Timothy K. Hart
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Membrane ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Cumene Hydroperoxide ◽  
Microscopic Analysis ◽  
Growth Media ◽  
Novikoff Hepatoma ◽  
Severe Hepatotoxicity ◽  
Transmission Electron

The insulin-sensitizing agent troglitazone (Rezulin) has been shown to cause severe hepatotoxicity and liver failure. While the toxic mechanism is still unknown, a component may be oxidative stress. We have demonstrated that increased lipid peroxidation, decreased glutathione levels, and collapse in mitochondrial membrane potential occurred following exposure of cultured hepatocytes to troglitazone. These hallmarks of oxidative stress can manifest morphologically in several ways including damage to cellular membranes, mitochondrial abnormalities, and cell death. The purpose of this study was to assess morphologic changes caused by exposure to troglitazone or cumene hydroperoxide (CH), a compound known to induce oxidative stress.HepG2 and Novikoff hepatoma cells were grown on poly-L-lysine coated 6-well plates overnight, and exposed to 10, 25, 50 or 100 μM concentrations of troglitazone or CH in the growth media for 2 hours. Cells were fixed with 2.5% glutaraldehyde in phosphate buffer, harvested, and pelleted. Pellets were post-fixed in 1% osmium tetroxide, and processed to epoxy resin.

scholarly journals Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study

2007 ◽  
Vol 18 (2) ◽  
pp. 153-157 ◽  
Author(s):  
Dimitrius Leonardo Pitol ◽  
Flavio Henrique Caetano ◽  
Laurelúcia Orive Lunardi
Keyword(s):  
Osmium Tetroxide ◽  
Microscopic Analysis ◽  
Electron Microscopic Analysis ◽  
Electron Microscopic ◽  
Cytochemical Study ◽  
Domestic Microwave Oven ◽  
Total Experiment Time ◽  
Transmission Electron ◽  
Rat Bone ◽  
Potassium Pyroantimonate

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4ºC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37ºC. In the first day, the specimens were irradiated 9 times and stored at 40ºC overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

An attempt to get an EEL filter image of biological sections using a spectrometer and a scanning device

1990 ◽  
Vol 48 (2) ◽  
pp. 22-23
Author(s):  
Yutaka Futaesaku ◽  
Kachiko Sekiya ◽  
Michiko Ono ◽  
Atsushi Nakamura ◽  
Yoshiko Nakamura ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Scanning Transmission Electron Microscope ◽  
Light Elements ◽  
Combined System ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Ultrathin Sections ◽  
Electron Energy Loss

An amount of light elements contained in biological specimens is barely a few, so that the detection of them in the ultrathin sections is imposible. An analysis of electron energy loss (EEL) filter image is an ideal to detect such as phosphorus including in the sections. CEM-902 is a commercially available electron microscope to use for a such purpose. The authers attempted to get EEL filter images using the combined system of a conventional scanning transmission electron microscope (STEM) with EELS analyzer.The cultured cells with calcified substances and the liver, kidney, thyroid gland were used for biological specimens. There were fixed with 2.5% glutaraldehyde and with or without 1% osmium tetroxide, and then embedded in epoxy resin. 40 run thick sections were analyzed without any staining and carbon coating using a Carl Zeiss CEM-902 or a JEOL 2000EX with a EELS analyzer and a Tracor-Northern TN-5500, and with a Gatan cryo-transfer holder.

scholarly journals Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload

2015 ◽  
Vol 308 (6) ◽  
pp. H651-H663 ◽  
Author(s):  
Danielle M. Yancey ◽  
Jason L. Guichard ◽  
Mustafa I. Ahmed ◽  
Lufang Zhou ◽  
Michael P. Murphy ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Mitochondrial Membrane ◽  
Volume Overload ◽  
Ros Production ◽  
Isolated Cardiomyocytes ◽  
Transmission Electron ◽  
Cyclical Stretch

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β2-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF.

Destruction of Actin Filaments by Osmium Tetroxide

1977 ◽  
Vol 35 ◽  
pp. 466-467
Author(s):  
P. Maupin-Szamier ◽  
T. D. Pollard
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Osmium Tetroxide ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Sodium Phosphate Buffer ◽  
Transmission Electron ◽  
The Difference ◽  
Rates Of Decay ◽  
Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Cell Reactivity Pattern of the Guinea Pig Cecal Mucosa Evidenced by the ZIO Technique

1982 ◽  
Vol 40 ◽  
pp. 50-51
Author(s):  
Juan Mora-Galindo ◽  
Jorge Arauz-Contreras
Keyword(s):  
Guinea Pig ◽  
Phase Contrast ◽  
Osmium Tetroxide ◽  
Cell Types ◽  
Cell Reactivity ◽  
Transmission Electron ◽  
Neural Tissues ◽  
Maturation Stages ◽  
Zinc Iodide ◽  
Cecal Mucosa

The zinc iodide-osmium tetroxide (ZIO) technique is presently employed to study both, neural and non neural tissues. Precipitates depends on cell types and possibly cell metabol ism as well.Guinea pig cecal mucosa, already known to be composed of epithelium with cells at different maturation stages and lamina propria which i s formed by morphologically and functionally heterogeneous cell population, was studied to determine the pat tern of ZIO impregnation. For this, adult Guinea pg cecal mucosa was fixed with buffered 1.2 5% g 1 utara 1 dehyde before incubation with ZIO for 16 hours, a t 4°C in the dark. Further steps involved a quick sample dehydration in graded ethanols, embedding in Epon 812 and sectioning to observe the unstained material under a phase contrast light microscope (LM) and a transmission electron microscope (TEM).

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

TEM comparison of osmium vs osmium with potassium ferricyanide secondary fixatives and the impact of secondary fixative temperature on tissue preservation/contrast quality

1996 ◽  
Vol 54 ◽  
pp. 910-911
Author(s):  
J. W. Horn ◽  
B. J. Dovey-Hartman ◽  
V. P. Meador
Keyword(s):  
Osmium Tetroxide ◽  
Potassium Ferricyanide ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Microscopic Evaluation ◽  
Cacodylate Buffer ◽  
Transmission Electron Microscopic ◽  
Glutaraldehyde Solution ◽  
Temperature Impact ◽  
The Impact

Osmium tetroxide (OsO4) is a universally used secondary fixative for routine transmission electron microscopic evaluation of biological specimens. Use of OsO4 results in good ultrastructural preservation and electron density but several factors, such as concentration, length of exposure, and temperature, impact overall results. Potassium ferricyanide, an additive used primarily in combination with OsO4, has mainly been used to enhance the contrast of lipids, glycogen, cell membranes, and membranous organelles. The purpose of this project was to compare the secondary fixative solutions, OsO4 vs. OsO4 with potassium ferricyanide, and secondary fixative temperature for determining which combination gives optimal ultrastructural fixation and enhanced organelle staining/contrast.Fresh rat liver samples were diced to ∼1 mm3 blocks, placed into porous processing capsules/baskets, preserved in buffered 2% formaldehyde/2.5% glutaraldehyde solution, and rinsed with 0.12 M cacodylate buffer (pH 7.2). Tissue processing capsules were separated (3 capsules/secondary fixative.solution) and secondarily fixed (table) for 90 minutes. Tissues were buffer rinsed, dehydrated with ascending concentrations of ethanol solutions, infiltrated, and embedded in epoxy resin.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

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