Micro- and Cryo-Techniques Prevent the Loss of Structural Information. EM-Studies on High-Pressure Frozen Tissues, Suspensions and Cell Monolayer.

1999 ◽  
Vol 5 (S2) ◽  
pp. 430-431
Author(s):  
H. Hohenberg
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Structural Information ◽  
Cell Monolayer ◽  
Freeze Substitution ◽  
Fine Needle Biopsy ◽  
Specimen Preparation ◽  
Optical Instruments ◽  
Cultivation Technique

Cells are information driven systems. Cellular information is stored in certain molecules, at certain places, in a certain concentration, at a particular time and under given physiological conditions. The goal of biological electron microscopy is to provide this information network, to correlate the cellular ultrastructure and its function. In this sense, it is essential to combine the high resolution of our electron optical instruments with a high information density of the biological system. Most of the structural information is lost in the course of the different preparation steps prior to electron microscopy. For this reason it is necessary that all preparation steps such as: 1. sampling: e.g. excision of tissues, 2. cryoimmobilisation, 3. follow-up procedures: e.g. freeze-fracturing, freeze-substitution and embedding, should have identical high quality levels preventing or minimizing the loss of structural information. To this aim our methodological activities focus on the development of special micro-techniques for the sampling of: a) native tissues, with an automatic fine-needle biopsy technique (1), of b) suspensions, with a special cellulose capillary technique (2), of c) cell monolayer, with a thin film cultivation technique (3) and the application/perfection of cryotechniques (high-pressure freezing (HPF) and freeze-substitution). In particular, the high-pressure freezer (HPM 010, Bal-Tec) has proven to be a highly useful tool for successful cryoimmobilization of almost any kinds of cells and tissues, bulk specimens (< 200 μm in thickness) being included. This freezing technique does not require any cryoprotection, and if combined with our micro-techniques the risk of inducing artefacts as a result of specimen preparation prior to freezing is minimized.

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.

Electron microscopy analysis of maize basal endosperm transfer cells processed by high-pressure freezing and freeze-substitution

2008 ◽  
Vol 14 (S2) ◽  
pp. 1502-1503
Author(s):  
B-H Kang ◽  
D Williams ◽  
K Kelley ◽  
K Backer-Kelley ◽  
P Chourey
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
New Mexico ◽  
Freeze Substitution ◽  
Transfer Cells ◽  
High Pressure Freezing ◽  
Endosperm Transfer Cells ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

The Connective Tissue Matrix of Cartilage as Revealed by Standard Fixation, Ruthenium Staining, High-Pressure Freezing, and Embedding in Water Soluble Media: A Comparison of Methods

1990 ◽  
Vol 48 (2) ◽  
pp. 326-327
Author(s):  
Douglas R. Keene
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Ruthenium Red ◽  
Water Soluble ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Microscopic Evaluation ◽  
Embedding Medium

Proteoglycan is a major component of the cartilage extracellular matrix, and the overall structure of this anionic molecule is highly dependent on the hydrated environment of cartilage. Without specific stabilization, proteoglycans are extracted or collapsed during deydration while processing for electron microscopy. The purpose of these experiments is to determine a method by which the structure of proteoglycans might be stabilized for electron microscopic evaluation.Chick sternal cartilage was prepared for transmission electron microscopy by the following methods and the resultant tissue ultrastructure compared: A) 1.5/1.5% gluteraldehyde/paraformaldehyde and 1% OsO4 fixation, dehydration in ethanol, propylene oxide, and embedding in Spurrs epoxy B) Fixation as in (A) directly followed by infiltration and embedding in Hexamethylol-melamine-methyl-ether (a water soluble embedding medium) trade name “nanoplast” C) Fixation by high pressure freezing followed by freeze substitution in acetone/OsO4 prior to embedding in epon 812. In variations of methods A and B above, ruthenium red (RR, 1500 ppm) or ruthenium hexamine trichloride (RHT, 6000 ppm) were added to the primary and secondary fixatives. All tissue sections were stained in uranyl acetate and lead citrate.

Ultrastructure of the host-parasite interaction in leaves of Duchesnea indica infected by the rust fungus Frommeëla mexicana var. indicae as revealed by high pressure freezing

2001 ◽  
Vol 79 (1) ◽  
pp. 49-57 ◽  
Author(s):  
C W Mims ◽  
C Rodriguez-Lother ◽  
E A Richardson
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Pressure ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Transfer Cells ◽  
Host Cells ◽  
High Pressure Freezing ◽  
Transmission Electron ◽  
Duchesnea Indica

A combination of scanning and transmission electron microscopy was used to examine the host-pathogen relationship in leaves of Duchesnea indica (Andrz) Focke infected by the rust fungus Frommeëla mexicana var. indicae McCain & Hennen. Samples for transmission electron microscopy were prepared using high pressure freezing followed by freeze substitution. This protocol provided excellent preservation of both host cells and fungal haustoria. Each haustorium of F. mexicana var. indicae possessed a long slender neck with a neck band and an expanded body that contained two nuclei positioned close together. The haustorial body was lobed and sometimes even branched but lacked septa. Details of the extrahaustorial membrane that separated each haustorium from the cytoplasm of its host cell were particularly well preserved. Extensive labyrinth cell wall ingrowths developed around haustorial necks, as well as elsewhere, in infected cells. These ingrowths appeared to be identical to those present in plant transfer cells. Transfer cells are thought to be involved in intensive solute transfer over short distances. This appears to be the first report of the development of transfer cells in response to infection by a plant pathogenic fungus.Key words: haustoria, transfer cells, freeze substitution, electron microscopy.

High Pressure Freezing has Come of Age - but is it Mature?

1999 ◽  
Vol 5 (S2) ◽  
pp. 426-427
Author(s):  
Kent McDonald
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Cell Types ◽  
Appropriate Technology ◽  
The Other ◽  
Current Status ◽  
Specimen Preparation ◽  
High Pressure Freezing ◽  
Upper Midwest ◽  
The U.S

It should be no secret by now that ultrarapid freezing is a superior method of specimen preparation for many biological EM projects, and that high pressure freezing is the most versatile of the freezing methods. While cryopreservation is not necessary for all EM studies, it is the method of choice for high resolution work and where “fixation artifacts”, such as distorted membranes, or extraction of the cytosol is a problem. It is true that the machines are expensive, and not always immediately available, but for some questions it is the appropriate technology to use. In the U.S., there are machines available for general use in the far West (Berkeley, CA), the upper Midwest (Madision, WI and Minneapolis, MN), and in the Northeast (Albany, NY). For locations of any of the other 8 machines around the country, interested users can call Technotrade, International at (603) 622-5011.Current Status of High Pressure Freezing: A decade ago, Studer et al. wrote an article entitled: “High Pressure Freezing Comes of Age” that illustrated how high pressure freezing (HPF) had become a proven technology, useful for preserving ultrastructure with unmatched fidelity in cell types that had previously been difficult to fix well for electron microscopy (EM).

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