High-Pressure Freezing and Freeze Substitution of In Vivo and In Vitro Cultured Plant Samples

2015 ◽  
pp. 117-134 ◽  
Author(s):  
Jose M. Seguí-Simarro
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Plant Samples

High-pressure freezing improves quantitative and qualitative structural data in the actinomycete microsymbiont frankia

1993 ◽  
Vol 51 ◽  
pp. 110-111
Author(s):  
R. Howard Berg
Keyword(s):  
High Pressure ◽  
Plant Root ◽  
Root Nodules ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Void Space ◽  
En Bloc ◽  
Tem Analysis

Symbiotic plant root nodules containing the nitrogen-fixing bacterium Frankia occur on a variety of woody shrubs and trees. Ever since the first micrographs of freeze substituted cells of Frankia in culture were published there has been impetus to see if freeze substitued nodule tissue will improve imaging of Frankia in vivo. High pressure freezing/freeze substitution (HPFS) accomplishes this.Frankia is an actinomycete that fixes N2 in a specialized multicellular, spherical structure termed the “symbiotic vesicle” that is surrounded by a multilamellate envelope (MLE) comprised of lipids. Early work based on MLE birefringence suggested the MLE was a O2 diffusion barrier, thereby protecting nitrogenase from O2- inactivation. Recently this has been challenged by freeze fracture data. Traditionally it has been assumed that the MLE is electrontranslucent because the lamina of the MLE are extracted by dehydration solvents, producing the “void space”--an extraction artifact hindering TEM analysis of MLE structure.En bloc staining with chromic acid stains the MLE, showing that the MLE is present after exposure to dehydration solvents and that the void space results from tissue shrinkage in the symbiotic vesicle (Figure 1).

scholarly journals Electron microscopy of plant samples by using high-pressure freezing/freeze substitution method

2019 ◽  
Vol 31 (1) ◽  
pp. 25-29
Author(s):  
Mayuko Sato ◽  
Mayumi Wakazaki ◽  
Yumi Goto ◽  
Kiminori Toyooka
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Substitution Method ◽  
Plant Samples

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.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

1990 ◽  
Vol 48 (3) ◽  
pp. 692-693
Author(s):  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Pathogenic Fungus ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Ice Crystals ◽  
High Pressure Freezing ◽  
Thin Walled Structures ◽  
Cytological Changes ◽  
Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

scholarly journals Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1597
Author(s):  
Thuong Thi Ho ◽  
Van Thi Pham ◽  
Tra Thi Nguyen ◽  
Vy Thai Trinh ◽  
Tram Vi ◽  
...  
Keyword(s):  
High Pressure ◽  
Neutralizing Antibodies ◽  
H5n1 Virus ◽  
Vaccine Development ◽  
Particle Characterization ◽  
Innovative Strategy ◽  
Specific Igg ◽  
Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

Ultrastructure of Sea Urchin Calcified Tissues after High-Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 131 (2) ◽  
pp. 116-125 ◽  
Author(s):  
Laurent Ameye ◽  
René Hermann ◽  
Philippe Dubois
Keyword(s):  
High Pressure ◽  
Sea Urchin ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Calcified Tissues

Vasopressin is metabolized by a trypsinlike enzyme in guinea pig amniotic fluid

1989 ◽  
Vol 257 (3) ◽  
pp. E354-E360 ◽  
Author(s):  
C. F. Uyehara ◽  
A. K. Sato ◽  
J. R. Claybaugh
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Guinea Pig ◽  
Amniotic Fluid ◽  
High Pressure Liquid Chromatography ◽  
Trypsin Inhibitor ◽  
Arginine Vasopressin ◽  
Proteolytic Enzyme

We have demonstrated that arginine vasopressin (AVP) is degraded to desglycinamide AVP by a trypsinlike enzyme found in guinea pig amniotic fluid. Incubation of [3H]AVP with guinea pig amniotic fluid in vivo or in vitro produced a metabolite that comigrated on high-pressure liquid chromatography with desglycinamide AVP in three different buffer systems. Also, AVP antisera that cross-reacted with standard desglycinamide AVP could detect this amniotic fluid metabolite. Because the enzyme responsible for the cleavage of glycinamide from AVP was likely to be trypsin, experiments with aprotinin, a trypsin inhibitor, were conducted. Results demonstrated that the production of the amniotic fluid AVP metabolite could be completely blocked in the presence of the trypsin inhibitor. In addition, examination of amniotic fluid collected from fetuses in the second half of gestation (term = 68 days) showed that AVP could not be metabolized to desglycinamide AVP until after 52 days of gestation. In conclusion, AVP appears to be metabolized by a trypsinlike enzyme in amniotic fluid, and because trypsin is a general proteolytic enzyme, the amniotic compartment may also serve as a clearance site for other proteins.

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