Life Sciences Commons^™

A New Versatile System For Freeze-Substitution, Freeze-Drying And Low Temperature Embedding Of Biological Specimens, H. Sitte, L. Edelmann, H. Hässig, H. Kleber, A. Lang

Scanning Microscopy

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 I Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN_2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. FS …

Improvements In Biological X-Ray Microanalysis: Cryoembedding For Specimen Preparation And Multivariate Statistical Analysis For Data Interpretation, C. Quintana, N. Bonnet

Scanning Microscopy

For biological X-ray microanalysis, cryoembedding (CE) combined with cryofixation (CF) and cryodehydration (CD) was already proposed as an alternative method to freeze-dried cryosections in 1984 by Wroblewski and Wroblewski. CD by freeze-drying (FD) is usually recommended because it provides better retention of diffusible elements. CD by freeze-substitution (FS) has the advantage of being simpler, giving more reproducible preservation of ultrastructure and causing fewer problems for resin infiltration. We have increased the retention of diffusible elements by using home-made devices for CS and CE in the new Lowicryl KllM and HM23 resins. These resins allow samples to be kept at a …

Optimization And Application Of Jet-Freezing, T. Müller, S. Moser, M. Vogt, C. Daugherty, M. V. Parthasarathy

Scanning Microscopy

Cryofixation is considered to be the best method for immobilizing biological material in its natural state. In jet-freezing, the specimen typically is sandwiched between two carriers and kept in place while a coolant is moved very rapidly against the opposite surfaces. The JFD 030 jet-freezing device has been used to optimize the operating parameters. The course of the temperature in place of a specimen was measured with thermocouples and recorded by an IBM-compatible personal computer using a specifically developed software program. Mean cooling rates, over the temperature range of 273K to 173K, achievable with different cryogens, including the non-flammable HCFC …

Scanning Electron Microscopy Of High-Pressure-Frozen Sea Urchin Embryos, Paul Walther, Ya Chen, Marek Malecki, Sara L. Steffen Zoran, Gerald P. Schatten, James B. Pawley

Scanning Microscopy

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was …

Comparison Of Cryopreparation Techniques For Electron Probe Microanalysis Of Cells As Exemplified By Human Erythrocytes, Karl Zierold

Scanning Microscopy

Erythrocytes in human blood were used to evaluate the reliability of cryopreparation techniques for electron probe X-ray microanalysis of biological cells and tissues. The elemental content determined by X-ray microanalysis of ultrathin freeze-dried cryosections was found to be consistent with data known from the literature. Considerable redistribution of the intracellular elemental composition was found after freeze-substitution as well as after freeze-drying followed by resin embedding. Two conclusions are drawn from this study: 1. Erythrocytes in human blood are a suitable reference specimen for evaluation of specimen preparation techniques for microanalysis. 2. At present, freeze-dried cryosections are the most reliable specimen …

Histochemical Demonstration And Microanalysis Of Possible Calcium Binding Sites In The Enamel Organ Of Rat Incisors, Y. Takano

Scanning Microscopy

The rat incisors obtained from rats perfused with high-calcium solution containing 30 to 50 mM CaCl₂ were processed for rapid freeze/freeze-substitution and embedded in epoxy resin. GBHA staining, a histochemical staining for calcium, of unhydrously prepared sections revealed a large number of granular Ca-GBHA reactions in the enamel organ, most of which being located along the lateral plasma membranes of the ameloblasts. In the ameloblast layer, the reaction was negative in the presecretory stage, became intense in concert with the onset of enamel matrix formation, and remained so by the end of the transitional stage where the reaction gradually …

Cryofixation Of Tissues For Electron Microscopy: A Review Of Plunge Cooling Methods, Keith P. Ryan

Scanning Microscopy

This review of cryofixation by plunge cooling surveys liquid coolants, plunge cooling devices, modeling and applications featuring time resolution. It then focuses on thermocouple experiments and ice crystal analyses. These highlight the effects of the design of specimen holders, the cold gas above the coolant which can freeze the specimen prematurely, specimen size, plunge velocity, coolant depth and ultimate coolant efficiencies. The ice crystal cavity analyses are validated by experiments which monitor the effects of high subzero temperatures on the storage of plunge cooled specimens and an experiment which monitored the rate of cryosubstitution. Ethane was found to be more …

Backscattered Electron Imaging For High Resolution Surface Scanning Electron Microscopy With A New Type Yag-Detector, Paul Walther, Rudolf Autrata, Ya Chen, James B. Pawley

Scanning Microscopy

Double Layer Coating for backscattered electron imaging is a coating and imaging method especially suitable for high resolution scanning electron microscopy (SEM) of large biological samples. Since the backscattered electron (BSE) signal from thin metal coating layers is quite low, field emission SEM's and very sensitive BSE-detectors are required for this method. In this study an improved BSE-detector of the YAG type was used with an in-lens type field emission SEM. Two samples were investigated in order to demonstrate and to improve the potential of this new approach: (1) cryo-prepared cultured kidney cells were shadowed by electron beam evaporation with …

Preservation And Immunogold Localization Of Lipids By Freeze-Substitution And Low Temperature Embedding, Wim Voorhout, Ida Van Genderen, Gerrit Van Meer, Hans Geuze

Scanning Microscopy

The success of postembedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level.

In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and …

Adsorption Staining Of Freeze-Substituted And Low Temperature Embedded Frog Skeletal Muscle With Cesium: A New Method For The Investigation Of Protein-Ion Interactions, L. Edelmann

Scanning Microscopy

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs⁺ -ions which are known to accumulate in living cells like K⁺ -ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be …

Aldehyde Fixation Causes Membrane Vesiculation During Platelet Exocytosis: A Freeze-Substitution Study, E. Morgenstern

Scanning Microscopy

Despite a plethora of reports on the ultrastructure of secretory granule release by exocytosis, the release of coagulant activity from stimulated platelets is still being attributed to membrane vesiculation. Membrane vesiculation and the formation of myelin figures have been shown to be artifacts of glutaraldehyde GA fixation. Cells fixed by direct osmium or rapid freezing are free of such structures. Yet there is still doubt that rapid freezing interferes with vesiculation process. This study has addressed this issue by examining: (1) whether freezing and freeze-substitution affects membrane vesiculation, (2) whether paraformaldehyde-fixation also induces the phenomenon, and (3) whether the aldehyde …

A Comparison Of Three Low Temperature Techniques Of Specimen Preparation For X-Ray Microanalysis, R. J. Condron, A. T. Marshall

Scanning Microscopy

A quantitative x-ray microanalytical comparison of intracellular ion concentrations in two tissues (chicken kidney and duck nasal salt gland), prepared as bulk frozen-hydrated, embedded freeze-dried and freeze-substituted samples, shows that there are similar losses of K⁺ and Pin freeze-dried and freeze-substituted samples in both types of tissue. It is suggested that this may be due to extraction by chloride-free Spurr's resin during infiltration. There was also an indication of an increase in Na⁺ concentration in freeze-dried samples.

In chicken kidney cells there was a reduction in Mg⁺⁺ and Ca⁺⁺ concentrations in freeze-dried and freeze-substituted samples and …

The Physical State Of Potassium In Frog Skeletal Muscle Studied By Ion-Sensitive Microelectrodes And By Electron Microscopy: Interpretation Of Seemingly Incompatible Results, L. Edelmann

Scanning Microscopy

According to the commonly accepted membrane pump theory most of cellular K⁺ ions are freely dissolved in free cellular water; the alternative association-induction hypothesis postulates that the bulk of cellular K⁺ is adsorbed (weakly bound) to cellular proteins which are maintained in a specific labile state in the cytoplasm of a living cell. K⁺ activities measured with ion-sensitive microelectrodes in the cytoplasm of frog skeletal muscle seem to confirm the claim that most of cellular K⁺ ions are free in cellular water. On the other hand, it is evident from electron microscopic ion binding studies that …

Olfactory And Nasal Respiratory Epithelia, And Foliate Taste Buds Visualized With Rapid-Freeze Freeze-Substitution And Lowicryl K11m Embedding. Ultrastructural And Initial Cytochemical Studies., Bert Ph. M. Menco

Scanning Microscopy

Rat olfactory and respiratory epithelia and Rhesus monkey taste buds were studied with rapid-freeze, acetone/0.1% uranyl acetate freeze-substitution and low-temperature Lowicryl K11M embedding, usually in the absence of other chemical fixation and cryoprotection procedures. Ultrastructural features of mucus, cytoplasm, including cytoskeletons, and membranes were better retained than with conventional methods. Some major examples: The mucus of the olfactory epithelium consisted of a single layer; that of the respiratory epithelium had an electron-opaque sol layer surrounding cilia and microvilli below a thin laminated electron-lucent gel layer. Taste-bud pores displayed a foam-like opaque secretory product, resembling the contents of secretory granules within …

X-Ray Microanalysis Of Calcium Containing Organelles In Resin Embedded Tissue, G. Nicaise, I. Gillot, A. K. Julliard, E. Keicher, S. Blaineau, J. Amsellem, J. C. Meyran, M. L. Hernandez-Nicaise, B. Ciapa, C. Gleyzal

Scanning Microscopy

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites : calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation.

Resin embedding is less demanding than cryomicrotomy and gives better …

The Cell Water Problem Posed By Electron Microscopic Studies Of Ion Binding In Muscle, L. Edelmann

Scanning Microscopy

The question whether cell K⁺ is free or bound in the striated frog muscle has been investigated during the last 10 years by different cryomethods and electron microscopy. The results support the view that most of cellular ions are osmotically inactive and that therefore the observed cell water activity must be explained by a model which assumes a specific cell water structure. According to the association-induction hypothesis, cell water is influenced by macromolecules and has low solubilities for Na⁺ and other solutes which therefore are partly excluded from cellular water. Autoradiography of frozen hydrated Na⁺ loaded muscles …

Ultrastructural Analysis Of Dynamic Cellular Processes: A Survey Of Current Problems, Pitfalls And Perspectives, Helmut Plattner, Gerd Knoll

Scanning Microscopy

Dynamic phenomena in cells that can be analyzed on the ultrastructural level comprise so different aspects as ion shifts, conformational changes of macromolecules, membrane particle rearrangements, lipid phase transitions, protein--protein interactions (notably ligand-receptor interactions, including their sorting and sequestration), reversible membrane-to-membrane contacts, membrane fusions, transcellular transport phenomena, restructuring of cytoskeletal elements, ciliary and flagellar beat, cell shape changes, etc. Only some of these phenomena can be analyzed under stationary conditions, while others are unidirectional and sometimes very rapid. Therefore, the methodical approaches to be used (primary methods and follow-up procedures) might be widely different. Quite different methods are available, such …

Low Temperature Techniques In Biomedical Microanalysis, Romuald Wróblewski, Joanna Wroblewski, Godfried M. Roomans

Scanning Microscopy

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation, or when many data from a large number of samples have to be acquired. Quantitative analysis is then usually carried out with the …