Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

PROTOPLASMA ◽  
1988 ◽  
Vol 146 (2-3) ◽  
pp. 143-149 ◽  
Author(s):  
R. W. Roberson ◽  
M. S. Fuller
Keyword(s):  
Freeze Substitution ◽  
Hyphal Tip

Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkorper, cytoskeleton and endomembranes after freeze-substitution

1981 ◽  
Vol 48 (1) ◽  
pp. 89-103
Author(s):  
R.J. Howard
Keyword(s):  
Freeze Substitution ◽  
Hyphal Growth ◽  
Coated Vesicle ◽  
Fusarium Acuminatum ◽  
Tip Cell ◽  
Transmission Electron ◽  
Cytoplasmic Vesicles ◽  
Cytoskeletal Elements ◽  
Tip Cells ◽  
Hyphal Tip

The ultrastructure of freeze-substituted tip cells of Fusarium acuminatum was analysed by conventional and high-voltage transmission electron microscopy (HVEM). At least 2 morphologically distinct types of Golgi-like endomembrane cisternae were observed, each existing as single, fenestrated sheets and tubular elements that were often very closely associated with mitochondria. From HVEM observations of thick (0.25 and 0.5 micron) sections, the Spitzenkorper appeared to correspond to an apical mass of vesicles. A network of microfilaments was identified among component vesicles of the Spitzenkorper and adjacent to developing septa. Microtubules were oriented primarily parallel to the direction of hyphal growth and were located in all areas of the cytoplasm, including the tip cell apex. Cytoplasmic vesicles were closely associated with these microtubules. From these observations it is suggested that cytoskeletal elements play important roles in localized cell wall formation. The filasome, a previously unreported type of coated vesicle in fungi, might also be involved in wall synthesis.

scholarly journals Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitution of hyphal tip cells.

1980 ◽  
Vol 87 (1) ◽  
pp. 55-64 ◽  
Author(s):  
R J Howard ◽  
J R Aist
Keyword(s):  
Intracellular Transport ◽  
Freeze Substitution ◽  
Ultrastructural Level ◽  
Spindle Pole ◽  
Voltage Electron ◽  
Spindle Pole Bodies ◽  
Fungal Morphogenesis ◽  
Cytoplasmic Microtubules ◽  
Tip Cells ◽  
Hyphal Tip

The effects of methyl benzimidazole-2-ylcarbamate (MBC), one of only a few agents that are active against microtubules of fungi, were analyzed at the ultrastructural level in freeze-substituted hyphal tip cells of Fusarium acuminatum. Nontreated and control cells had numerous microtubules throughout. After just 10 min of exposure to MBC, almost no cytoplasmic microtubules were present, except near spindle pole bodies. After 45 min of exposure to MBC, no microtubules were present in hyphal tip cells, but they were present in the relatively quiescent subapical cells. These observations suggested that there are different rates of turnover for cytoplasmic microtubules in apical and subapical cells and for microtubules near spindle pole bodies and that MBC acts by inhibiting microtubules assembly. A statistical analysis of the distribution of intracytoplasmic vesicles in thick sections of cells treated with MBC, D2O or MBC + D2O was obtained by use of a high-voltage electron microscope. More than 50% of the vesicles in the apical 30 micrometers of control cells were found to lie within 2 micrometers of the tip cell apex. MBC treatment caused this vesicle distribution to become uniform, resulting in a substantial increase in the number of vesicles in subapical regions. The reduction in the number of cytoplasmic microtubules, induced by MBC, apparently inhibited intracellular transport of these vesicles and rendered random the longitudinal orientation of mitochondria. In most cases, D2O appeared capable of preventing these MBC-effects through stabilization of microtubules. These observations support the "vesicle hypothesis" of tip growth and establish a transport role for cytoplasmic microtubules in fungal morphogenesis.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

Electron microscopic studies on ultrathin frozen sections of lactating mouse mammary gland

1978 ◽  
Vol 36 (2) ◽  
pp. 46-47
Author(s):  
Jan Zarzycki ◽  
Joseph Szroeder
Keyword(s):  
Mammary Gland ◽  
Protein Denaturation ◽  
Chemical Constituents ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Mouse Mammary Gland ◽  
Electron Microscopic ◽  
Preparation Methods ◽  
Microscopic Studies ◽  
Ultrathin Frozen Sections

The mammary gland ultrastructure in various functional states is the object of our investigations. The material prepared for electron microscopic examination by the conventional chemical methods has several limitations, the most important are the protein denaturation processes and the loss of large amounts of chemical constituents from the cells. In relevance to this,one can't be sure about a degree the observed images are adequate to the realy ultrastructure of a living cell. To avoid the disadvantages of the chemical preparation methods,some autors worked out alternative physical methods based on tissue freezing / freeze-drying, freeze-substitution, freeze-eatching techniqs/; actually the technique of cryoultraraicrotomy,i,e.cutting ultrathin sections from deep frozen specimens is assented as a complete alternative method. According to the limitations of the routine plastic embbeding methods we were interested to analize the mammary gland ultrastructure during lactation by the cryoultramicrotomy method.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Techniques for cryoultramicrotomy of propane jet frozen biological samples

1986 ◽  
Vol 44 ◽  
pp. 260-261
Author(s):  
B. Craig ◽  
L. Hawkey ◽  
A. LeFurgey
Keyword(s):  
Freeze Fracture ◽  
Freeze Substitution ◽  
Heart Cells ◽  
Crystal Formation ◽  
X Ray ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
A New Technique

Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

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