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 …

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 Quick-Freezing Of Single Intact Skeletal Muscle Fibers At Known Time Intervals Following Electrical Stimulation, Rashid Nassar, Nancy R. Wallace, Isaiah Taylor, Joachim R. Sommer

Scanning Electron Microscopy

Single intact frog skeletal muscle fibers quick-frozen after known time intervals following electrical stimulation are examined electron microscopically in thin sections, after freeze-substitution, in freeze-fracture/etch preparations, and in cryosections prepared for x-ray microprobe analysis. Techniques are described to perform these operations on a single fiber. Notable morphological differences between conventionally fixed and cryopreserved muscle fibers, and between fibers quick- frozen at different post-stimulation intervals are demonstrated.

Freeze-Drying And Related Preparation Techniques For Biological Microprobe Analysis, Romuald Wróblewski, Joanna Wróblewski, Matti Anniko, Lars Edström

Scanning Electron Microscopy

An X-ray microanalytical and morphological investigation has been carried out on rapidly frozen, freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze-substitution and freeze-drying. The investigation also included an analysis of specimens infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl HM20. The morphological preservation of Lowicryl embedded tissue was adequate for the identification of different cell structures like nuclei, mitochondria, lysosomes and different types of endoplasmic reticulum. X-ray microanalytical investigation of low temperature embedded material displayed an elemental composition of cells and organelles similar to that found in freeze-dried …

Two Opposing Theories Of The Cell: Experimental Testing By Cryomethods And Electron Microscopy, L. Edelmann

Scanning Electron Microscopy

A main controversial issue in cell biology concerns the molecular mechanism responsible for K⁺ accumulation in living cells and Na⁺ exclusion from them. The alternative theoretical descriptions of these phenomena are based on different assumptions about the physical state of cellular Na⁺, K⁺ and H₂O. In this article it is shown with striated muscles how cryomethods and microanlytical electron microscopy may be used to test the opposing theories. It is concluded that these methods may yield more realistic informations about the physical state of cellular K⁺ and Na⁺ than measurements with …

Low Temperature Embedding, E. Carlemalm, W. Villiger, J. -D. Acetarin, E. Kellenberger

Scanning Electron Microscopy

The Lowicryl resins K4M and HM20 are methacrylate/acrylate based formulations which can re used for embedding biological material at low temperature in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. The resins are applicable over a very extended temperature range, approximately 210°K to 340°K. Even lower temperatures down to ca. 190°K can be reached with two new resins, K11M and HM23. Test embeddings of unfixed material after freeze-substitution have given promising results which could re useful for imnunocytochemical labeling. Lipid extraction is small or absent when the two new resins are used in combination with …

Freeze Substitution And Low Temperature Embedding, B. Humbel, M. Müller

Scanning Electron Microscopy

The problems of conventional EM-preparation techniques based on chemical fixation may be overcome to a considerable extent by freeze substitution techniques. Although at present substitution cannot be performed at sufficiently low temperatures to prevent the recrystallization of vitrified aqueous specimens, thin sectioned biological samples show an improved information density. If freeze-substitution is combined with conventional embedding above 273 K (Epon/Araldite, Spurr's resin) the substituting organic solvent must contain stabilizing agents such as osmiumtetroxide, glutaraldehyde or uranylions. In combination with low temperature embedding procedures (Lowicryl) completely unfixed samples are obtained, which are suitable for immunolabelling and electron spectroscopic experiments. Water in …