Improved structural preservation in freeze-substituted sporidia ofUstilago avenae?a comparison with low-temperature embedding

PROTOPLASMA ◽  
1986 ◽  
Vol 135 (2-3) ◽  
pp. 181-181
Author(s):  
S. Hippe ◽  
M. Hermanns
Keyword(s):  
Low Temperature ◽  
Structural Preservation

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

scholarly journals Immunostaining of keratin and vimentin in epidermis: comparison of different post-embedding immunogold techniques for electron microscopy.

1989 ◽  
Vol 37 (6) ◽  
pp. 863-868 ◽  
Author(s):  
G Mahrle ◽  
H J Schulze ◽  
A Kuhn ◽  
A Wevers
Keyword(s):  
Low Temperature ◽  
Polyclonal Antibodies ◽  
Cluster Formation ◽  
Linear Distribution ◽  
Immunogold Staining ◽  
Fixed Material ◽  
Staining Methods ◽  
Linear Alignment ◽  
Structural Preservation ◽  
Lowicryl K4m

The present study compares different post-embedding staining methods, including conventional and low-temperature embedding techniques, for demonstration of the keratin and vimentin cytoskeleton of epidermal cells, applying commercially available polyclonal and monoclonal antibodies. Immunogold staining (5-nm particles) was performed on the following material: (a) osmium-fixed and Durcupan-embedded material, etched with various solutions; (b) aldehyde-fixed material embedded in Lowicryl K4M at 4 degrees C and -35 degrees C; (c) aldehyde-fixed material embedded in Lowicryl K11M at -60 degrees C with and without cryoprotection with glycerol. In conventionally embedded material we failed to demonstrate intermediate filaments, whereas they were stained after low-temperature embedding with Lowicryl, i.e., K4M 4 degrees C, K4M -35 degrees C, and K11M -60 degrees C. The keratin and vimentin cytoskeleton reacted exclusively with polyclonal antibodies. The best results for antigenicity as well as structural preservation were achieved by post-embedding staining of K4M -35 degrees C-embedded material. Antibodies to keratin stained the cytoskeleton in keratinocytes of all epidermal layers. Filaments were decorated in a linear alignment. Antibodies to vimentin stained the cytoskeleton of Langerhans cells and melanocytes. In these cells a linear distribution pattern of the reaction product along the filaments and an extrafilamentous cluster formation were observed, indicating staining of vimentin and a vimentin-associated protein.

Structural preservation and absolute contrast of catalase crystal sections prepared with tannic acid

1983 ◽  
Vol 41 ◽  
pp. 448-449
Author(s):  
C.W. Akey ◽  
M. Szalay ◽  
S.J. Edelstein
Keyword(s):  
Tannic Acid ◽  
Beef Heart ◽  
X Rays ◽  
Screw Axis ◽  
General Applicability ◽  
Thin Sections ◽  
Beef Liver ◽  
3 Dimensional ◽  
Dimensional Reconstruction ◽  
Structural Preservation

Three methods of obtaining 20 Å resolution in sectioned protein crystals have recently been described. They include tannic acid fixation, low temperature embedding and grid sectioning. To be useful for 3-dimensional reconstruction thin sections must possess suitable resolution, structural fidelity and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of crystals of Pseudomonas cytochrome oxidase, orthorhombic beef liver catalase and beef heart F1-ATPase. In order to develop methods with general applicability, we have concentrated our efforts on a trigonal modification of catalase which routinely demonstrated a resolution of 40 Å. The catalase system is particularly useful since a comparison with the structure recently solved with x-rays will permit evaluation of the accuracy of 3-D reconstructions of sectioned crystals.Initially, we re-evaluated the packing of trigonal catalase crystals studied by Longley. Images of the (001) plane are of particular interest since they give a projection down the 31-screw axis in space group P3121. Images obtained by the method of Longley or by tannic acid fixation are negatively contrasted since control experiments with orthorhombic catalase plates yield negatively stained specimens with conditions used for the larger trigonal crystals.

Exsolution and inversion in pigeonite from the Whin Sill, Northern England

1978 ◽  
Vol 36 (1) ◽  
pp. 474-475
Author(s):  
P.P.K. Smith
Keyword(s):  
High Temperature ◽  
Low Temperature ◽  
Homogeneous Nucleation ◽  
Silicate Mineral ◽  
Antiphase Boundaries ◽  
Two Phase ◽  
Primitive Form ◽  
The Core ◽  
Nucleation Mechanisms ◽  
And Inversion

Grains of pigeonite, a calcium-poor silicate mineral of the pyroxene group, from the Whin Sill dolerite have been ion-thinned and examined by TEM. The pigeonite is strongly zoned chemically from the composition Wo8En64FS28 in the core to Wo13En34FS53 at the rim. Two phase transformations have occurred during the cooling of this pigeonite:- exsolution of augite, a more calcic pyroxene, and inversion of the pigeonite from the high- temperature C face-centred form to the low-temperature primitive form, with the formation of antiphase boundaries (APB's). Different sequences of these exsolution and inversion reactions, together with different nucleation mechanisms of the augite, have created three distinct microstructures depending on the position in the grain.In the core of the grains small platelets of augite about 0.02μm thick have farmed parallel to the (001) plane (Fig. 1). These are thought to have exsolved by homogeneous nucleation. Subsequently the inversion of the pigeonite has led to the creation of APB's.

Low temperature x-ray microanalysis of frozen-hydrated tobacco leaves

1984 ◽  
Vol 42 ◽  
pp. 284-285
Author(s):  
S. Edith Taylor ◽  
Patrick Echlin ◽  
May McKoon ◽  
Thomas L. Hayes
Keyword(s):  
Low Temperature ◽  
Lemna Minor ◽  
Root Tips ◽  
Tobacco Leaves ◽  
X Ray ◽  
Whole Cells ◽  
Carbon Coated ◽  
The Right ◽  
Beam Currents ◽  
The University

Low temperature x-ray microanalysis (LTXM) of solid biological materials has been documented for Lemna minor L. root tips. This discussion will be limited to a demonstration of LTXM for measuring relative elemental distributions of P,S,Cl and K species within whole cells of tobacco leaves.Mature Wisconsin-38 tobacco was grown in the greenhouse at the University of California, Berkeley and picked daily from the mid-stalk position (leaf #9). The tissue was excised from the right of the mid rib and rapidly frozen in liquid nitrogen slush. It was then placed into an Amray biochamber and maintained at 103K. Fracture faces of the tissue were prepared and carbon-coated in the biochamber. The prepared sample was transferred from the biochamber to the Amray 1000A SEM equipped with a cold stage to maintain low temperatures at 103K. Analyses were performed using a tungsten source with accelerating voltages of 17.5 to 20 KV and beam currents from 1-2nA.

Bulk standards for low-temperature biological x-ray microanalysis

1984 ◽  
Vol 42 ◽  
pp. 282-283
Author(s):  
P. Echlin ◽  
M. McKoon ◽  
E.S. Taylor ◽  
C.E. Thomas ◽  
K.L. Maloney ◽  
...  
Keyword(s):  
Phase Separation ◽  
Low Temperature ◽  
Analytical Method ◽  
Salt Solutions ◽  
Absolute Concentration ◽  
Cooling Process ◽  
X Ray ◽  
The Absolute ◽  
Analytical Procedures ◽  
Electrical Conductors

Although sections of frozen salt solutions have been used as standards for x-ray microanalysis, such solutions are less useful when analysed in the bulk form. They are poor thermal and electrical conductors and severe phase separation occurs during the cooling process. Following a suggestion by Whitecross et al we have made up a series of salt solutions containing a small amount of graphite to improve the sample conductivity. In addition, we have incorporated a polymer to ensure the formation of microcrystalline ice and a consequent homogenity of salt dispersion within the frozen matrix. The mixtures have been used to standardize the analytical procedures applied to frozen hydrated bulk specimens based on the peak/background analytical method and to measure the absolute concentration of elements in developing roots.

Field ion microscopy

1988 ◽  
Vol 46 ◽  
pp. 434-435
Author(s):  
Gert Ehrlich
Keyword(s):  
Low Temperature ◽  
Sample Surface ◽  
Low Pressure ◽  
Radius Of Curvature ◽  
Field Ion Microscopy ◽  
Phosphor Screen ◽  
Ion Microscopy ◽  
Field Ion Microscope

The field ion microscope, devised by Erwin Muller in the 1950's, was the first instrument to depict the structure of surfaces in atomic detail. An FIM image of a (111) plane of tungsten (Fig.l) is typical of what can be done by this microscope: for this small plane, every atom, at a separation of 4.48Å from its neighbors in the plane, is revealed. The image of the plane is highly enlarged, as it is projected on a phosphor screen with a radius of curvature more than a million times that of the sample. Müller achieved the resolution necessary to reveal individual atoms by imaging with ions, accommodated to the object at a low temperature. The ions are created at the sample surface by ionization of an inert image gas (usually helium), present at a low pressure (< 1 mTorr). at fields on the order of 4V/Å.

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