Ultrastructure of Frozen Thin Sections of Skeletal Muscle

1972 ◽  
Vol 30 ◽  
pp. 304-305
Author(s):  
Richard S. Demaree ◽  
Terrance A. O'Donnell
Keyword(s):  
Skeletal Muscle ◽  
Liquid Nitrogen ◽  
Cutting Speed ◽  
Ice Crystals ◽  
Knife Edge ◽  
Fresh Tissue ◽  
Thin Sections ◽  
Clearance Angle ◽  
Cryoprotective Agents ◽  
Thin Sectioning

Reliable equipment has just recently become available which permits thin-sectioning of frozen specimens. We have been studying the effects of diet and exercise on skeletal muscle and are using cryoultramicrotomy as a check on fixation, dehydration, and embedding procedures.Canine triceps or pectineus muscles were obtained using either surgical or needle biopsy techniques. Fresh tissue was diced into 1mm cubes and either frozen fresh or fixed in 2.5% phosphate-buffered glutaraldehyde (5, 10, 15 or 30 minutes). Freezing by immersing specimens in liquid nitrogen consistently produced many ice crystals. However, freezing specimens by immersing them in isopentane cooled with liquid nitrogen produced fewer ice crystals. Cryoprotective agents, such as glycerine, yielded no appreciable improvement in preservation.Unfixed, frozen tissue was thin-sectioned with the specimen at -90° C, the knife at -30° C, clearance angle 5°, knife edge 55°, at a cutting speed of 2 mm/sec. Fixed, frozen muscle was thin-sectioned at the same temperatures with a knife clearance angle of 7°, knife edge 40°, at a cutting speed of lOmm/sec.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

On the Location of Stain in Mitochondria

1968 ◽  
Vol 26 ◽  
pp. 128-129
Author(s):  
G. B. Haydon ◽  
S. O. Smith
Keyword(s):  
Skeletal Muscle ◽  
Heavy Metal ◽  
Oxidative Phosphorylation ◽  
Focal Plane ◽  
Uranyl Acetate ◽  
Thick Section ◽  
Lead Citrate ◽  
Thin Sections ◽  
Thin Sectioning ◽  
Acetate Lead

The degree of matrical staining in isolated and tissue mitochondria has been shown to be a function of their state of oxidative-phosphorylation at the time of fixation (J. Histochem. Cytochem. 15:752, 1967). High resolution EM in conjunction with these studies revealed amplitude contrast images 10 AU and larger in diameter which are independent of the focal plane. These amplitude densities have been interpreted as individual aggregates of heavy metal stain (J. Cell Biol. 35:55A, 1967). Two approaches have been used in an effort to locate such stain aggregates.(1) Four micron epoxy sections of skeletal muscle, cardiac muscle, and liver were stained with uranyl acetate, lead citrate, or both, following the methods used to stain 500 AU sections for EM. After staining, the thick epon sections were re-embedded and thin sections cut perpendicular to the stained surface. Either or both of these metal stains penetrate 500 to 1000 AU into the thick section and give the tissue increased contrast similar to that due to staining after thin sectioning. The mitochondrial matrices are lightly stained indicating that they are not in a state of active oxidative-phosphorylation. Figure 1 shows such mitochondria at low magnification before (Figure 1a) and after (Figure lb) restaining with uranyl acetate and lead citrate. After restaining it is no longer possible to identify the original stained surface.

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.

Structures Formed by Cryoprotective Agents Used in Freezc-Etching

1971 ◽  
Vol 29 ◽  
pp. 448-449
Author(s):  
G. G. Cocks ◽  
C. E. Cluthe
Keyword(s):  
Aqueous Solution ◽  
Polyethylene Oxide ◽  
Liquid Nitrogen Temperature ◽  
Ice Crystals ◽  
Etching Technique ◽  
Structural Constituent ◽  
Cryoprotective Agents ◽  
Quick Freezing ◽  
Freeze Etching ◽  
Fractured Surface

The freeze etching technique is potentially useful for examining dilute solutions or suspensions of macromolecular materials. Quick freezing of aqueous solutions in Freon or propane at or near liquid nitrogen temperature produces relatively large ice crystals and these crystals may damage the structures to be examined. Cryoprotective agents may reduce damage to the specimem, hut their use often results in the formation of a different set of specimem artifacts.In a study of the structure of polyethylene oxide gels glycerol and sucrose were used as cryoprotective agents. The experiments reported here show some of the structures which can appear when these cryoprotective agents are used.Figure 1 shows a fractured surface of a frozen 25% aqueous solution of sucrose. The branches of dendritic ice crystals surrounded hy ice-sucrose eutectic can be seen. When this fractured surface is etched the ice in the dendrites sublimes giving the type of structure shown in Figure 2. The ice-sucrose eutectic etches much more slowly. It is the smooth continuous structural constituent surrounding the branches of the dendrites.

Ferritin Labeled Antibody Staining of Thin Sections

1969 ◽  
Vol 27 ◽  
pp. 420-421
Author(s):  
J. D. McLean ◽  
S. J. Singer
Keyword(s):  
Biological Material ◽  
Water Soluble ◽  
Thin Sections ◽  
Antibody Staining ◽  
Thin Sectioning ◽  
Ultrathin Sections

The successful application of ferritin labeled antibodies (F-A) to ultrathin sections of biological material has been hampered by two main difficulties. Firstly the normally used procedures for the preparation of material for thin sectioning often result in a loss of antigenicity. Secondly the polymers employed for embedding may non-specifically absorb the F-A. Our earlier use of cross-linked polyampholytes as embedding media partially overcame these problems. However the water-soluble monomers used for this method still extract many lipids from the material.

scholarly journals ULTRASTRUCTURE AND CALCIUM TRANSPORT IN CRUSTACEAN MUSCLE MICROSOMES

1971 ◽  
Vol 48 (1) ◽  
pp. 49-60 ◽  
Author(s):  
R. J. Baskin
Keyword(s):  
Freeze Drying ◽  
Calcium Uptake ◽  
Membrane Thickness ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Crustacean Muscle ◽  
Thin Sectioning ◽  
Critical Point Drying ◽  
Freeze Etching ◽  
Microscopic Techniques

Fragmented sarcoplasmic reticulum (FSR) from crustacean muscle was examined following preparation by a variety of electron microscopic techniques. The 30–40 A particles which appeared on the outer surface of FSR vesicles following negative staining were not observed following preparation by freeze-drying, freeze-etching, thin sectioning, or critical-point drying. Crustacean FSR exhibited high values of calcium uptake and extensive nodular formation in the presence of oxalate. 80–90 A diameter membrane particles were seen in freeze-etch preparations of both intact lobster muscle and FSR vesicles. Thin sections of FSR vesicles revealed a membrane thickness of 60–70 A. The membrane appeared to be triple layered, each layer having a thickness of 20–25 A.

scholarly journals Connectin filaments in stretched skinned fibers of frog skeletal muscle.

1984 ◽  
Vol 99 (4) ◽  
pp. 1391-1397 ◽  
Author(s):  
K Maruyama ◽  
H Sawada ◽  
S Kimura ◽  
K Ohashi ◽  
H Higuchi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscopic ◽  
Thin Filaments ◽  
Thin Sections ◽  
Cytoskeletal Structure ◽  
Present Information ◽  
Sds Page ◽  
Myosin Filaments ◽  
Elastic Protein ◽  
Passive Stretch

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

scholarly journals MAST CELL STAIN FOR HISTAMINE IN FREEZE-DRIED EMBEDDED TISSUE

1968 ◽  
Vol 16 (6) ◽  
pp. 433-439 ◽  
Author(s):  
WALTER B. SHELLEY ◽  
SVEN ÖHMAN ◽  
HERBERT M. PARNES
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Fluorescence Microscopy ◽  
Liquid Nitrogen ◽  
New Method ◽  
Polyethylene Glycols ◽  
Ethyl Benzene ◽  
Fresh Tissue ◽  
Bright Yellow ◽  
Freeze Dried

A new method is described which permits staining of the histamine in mast cells of embedded tissue. Small specimens of fresh tissue, rapidly frozen in isopentane-liquid nitrogen, are dried in vacuo at –35°C, embedded in paraffin, sectioned and stained with o-phthalaldehyde in ethyl benzene. Mast cell histamine appears bright yellow as viewed under fluorescence microscopy. The procedure is standardized, suitable for a wide variety of tissues and remarkably free of diffusion artifacts. The uncut blocks may be stored for months with no changes in later histamine-staining potential. Alternatively, embedding may be done in polyethylene glycols with satisfactory results.

scholarly journals MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

1974 ◽  
Vol 60 (3) ◽  
pp. 653-663 ◽  
Author(s):  
Akitsugu Saito ◽  
Murray Smigel ◽  
Sidney Fleischer
Keyword(s):  
Skeletal Muscle ◽  
Cross Sections ◽  
Rat Kidney ◽  
Respiratory Control ◽  
Heart Tissue ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Fresh Tissue ◽  
Isolated Mitochondria ◽  
Mammalian Tissues

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.

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