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.

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.

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.

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).

Preservation of Biomembranes by High Pressure Freezing ?

1999 ◽  
Vol 5 (S2) ◽  
pp. 428-429
Author(s):  
Martin Müller ◽  
Jens Listemann ◽  
Eyal Shimony ◽  
Paul Walther
Keyword(s):  
High Pressure ◽  
Structural Information ◽  
Ambient Pressure ◽  
High Time Resolution ◽  
Specimen Preparation ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cellular Events ◽  
Preparation Techniques

Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph.Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1).High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.

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.

Cryosectioning plant roots prepared by high-pressure freezing

1987 ◽  
Vol 45 ◽  
pp. 662-663
Author(s):  
R.E. Crang ◽  
M. Mueller ◽  
K. Zierold
Keyword(s):  
High Pressure ◽  
Cell Walls ◽  
Plant Tissues ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Vitreous State ◽  
Radial Depth ◽  
Irregular Topography ◽  
Considerable Depth ◽  
Conventional Freezing

Obtaining frozen-hydrated sections of plant tissues for electron microscopy and microanalysis has been considered difficult, if not impossible, due primarily to the considerable depth of effective freezing in the tissues which would be required. The greatest depth of vitreous freezing is generally considered to be only 15-20 μm in animal specimens. Plant cells are often much larger in diameter and, if several cells are required to be intact, ice crystal damage can be expected to be so severe as to prevent successful cryoultramicrotomy. The very nature of cell walls, intercellular air spaces, irregular topography, and large vacuoles often make it impractical to use immersion, metal-mirror, or jet freezing techniques for botanical material.However, it has been proposed that high-pressure freezing (HPF) may offer an alternative to the more conventional freezing techniques, inasmuch as non-cryoprotected specimens may be frozen in a vitreous, or near-vitreous state, to a radial depth of at least 0.5 mm.

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).

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.

Sign in / Sign up

Export Citation Format

Share Document