scholarly journals Ultrastructural analysis of single-membrane-bound organelles in plant cells: A comparison of high-pressure freezing versus chemical fixation

2013 ◽  
Vol 25 (1) ◽  
pp. 11-14 ◽  
Author(s):  
Chieko Saito
Keyword(s):  
High Pressure ◽  
Plant Cells ◽  
Ultrastructural Analysis ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Single Membrane ◽  
Membrane Bound

Jet freezing of cells and tissues with and without cryoprotectants

1993 ◽  
Vol 51 ◽  
pp. 106-107
Author(s):  
M.V. Parthasarathy ◽  
Carole Daugherty ◽  
T. Müller
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
Plant Cells ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
High Quality ◽  
The Past ◽  
Copper Specimen ◽  
Cell Structures

For the past several years cryofixation/freeze-substitution techniques have become valuable alternatives to chemical fixation of biological specimens. The superiority of cryofixation in preserving labile cell structures has been documented in several studies. Commercially available jet freezers and the BAL-TEC HPM010 high pressure freezer have extended high quality cryofixation from monolayer cells to cells relatively deep inside tissues. High pressure freezing can theoretically freeze biological materials of 0.5 mm thickness without the use of cryoprotectants and propane jet freezing is reported to freeze biological samples up to 40 μm in thickness without cryoprotection. Although high pressure freezing is the obvious method of choice for freezing large biological samples, its high cost combined with its apparent inability to consistently preserve microfilaments in some plant cells has prompted us to explore the capability of jet freezing to yield well frozen samples with and without cryoprotectants.We used the commercially available jet freezer JFD 030 (BAL-TEC) to obtain our results. Tightly pelleted cells sandwiched between 0.1 mm thick copper specimen carriers normally froze well without any cryoprotectants, after propane jet freezing (Figs. 1-2).

Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes

Nematology ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 103-110
Author(s):  
Myriam Claeys ◽  
Nurul Dwi Handayani ◽  
Vladimir V. Yushin ◽  
Prabowo Lestari ◽  
Antarjo Dikin ◽  
...  
Keyword(s):  
High Pressure ◽  
Fast Rate ◽  
Low Cost ◽  
Heterodera Schachtii ◽  
Fixation Method ◽  
Rapid Freezing ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Cyst Nematodes ◽  
Second Stage

Summary Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.

Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

2001 ◽  
Vol 7 (S2) ◽  
pp. 728-729
Author(s):  
Paul Walther
Keyword(s):  
High Pressure ◽  
Physiological State ◽  
Electron Microscopic Observation ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Freeze Fracturing

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

Cryofixation of various biological samples by high pressure freezing

1992 ◽  
Vol 50 (1) ◽  
pp. 752-753
Author(s):  
LUCY RU-SIU YIN
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
High Rate ◽  
Crystal Formation ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Native State ◽  
Biological Specimen ◽  
Ice Crystal Formation

The ultimate aim of ultrastructural fixation of biological specimen is to preserve all the compartments in their native state. Cryofixation is a superior method than conventional chemical fixation in reaching this goal. However, ice crystal formation during cryofixation often damages the structures. High pressure (2100 bar) freezing provides a way to alter freezing properties while cool down the specimen at a relatively high rate, minimizing the ice crystal formation. Nearly vitrified samples(up to 500 um) have been obtained with this method. Samples in suspension tend to get lost during high pressure freezing. The low percentage (∼30%) of successfully cryofixed specimens can be improved if the sample completely fills the cavity of the metal specimen carriers in which the specimen is frozen. Various methods to overcome sample loss are reported in this study.

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