Karl Zierold

Cryotechniques in biological electron microscopy

I Fundamentals.- 1 Physics of Water and Ice: Implications for Cryofixation.- 1 Introduction.- 2 Functions of Water in Cryospecimens.- 3 Water Below Room Temperature.- 4 Aqueous Solutions Below Room Temperature.- 5 Specimen Cooling.- References.- 2 The Response of Biological Macromolecules and Supramolecular Structures to the Physics of Specimen Cryopreparation.- 1 Introduction.- 2 The Hydration Shells of Biological Macromolecules and Supramolecular Structures.- 3 Phases and Interface Phenomena.- 4 The Particular Case of the Biological Membrane.- 5 A Potpourri of Collapses.- References.- 3 Electron Beam Radiation Damage to Organic and Biological Cryospecimens.- 1 Introduction.- 2 Electron Beam/Specimen Interaction.- 3 Radiation Damage to Organic Materials at Low Temperature.- 4 Radiation Damage to Ice.- 5 Radiation Damage to Frozen-Hydrated and Vitrified-Hydrated Specimens.- 6 Conclusions.- References.- II General Methodology.- 4 Cryofixation Without Pretreatment at Ambient Pressure.- 1 Introduction.- 2 Cryofixation with Liquid Cryogen.- 3 Impact Cryofixation (Slamming).- 4 Discussion.- References.- 5 Cryoeleetron Microscopy of Vitrified Specimens.- 1 Introduction.- 2 Vitrification.- 3 Preparation of Thin Specimens.- 4 Image Formation.- 5 Beam Damage.- References.- 6 Cryoultramicrotomy.- 1 Introduction.- 2 Technical Aspects of Cryoultramicrotomy.- 3 Physical Aspects of Cryoultramicrotomy.- 4 Conclusions.- References.- 7 Freeze-Substitution and Freeze-Drying.- 1 Introduction.- 2 Methodology: Theoretical and Experimental Data.- 3 Procedures and Instrumentation.- 4 Critical Evaluation.- References.- III Special Aspects.- 8 Theory and Practice of High Pressure Freezing.- 1 Introduction.- 2 Freezing Under Atmospheric Pressure.- 3 Freezing Under High Pressure.- 4 The Main Practical Problems of Pressure-Freezing.- 5 The High Pressure Freezing Machine (Balzers HPM 010).- 6 Some Practical Advice.- 7 Discussion of Results.- References.- 9 Freeze-Etching of Dispersions, Emulsions and Macromolecular Solutions of Biological Interest 192.- 1 Introduction.- 2 Specific Problems of Specimen Preparation.- 3 Determination of Particle Concentrations and Molecular Weights.- 4 Measurements of Size and Shape.- 5 Structure of Dispersions in Bulk and at Interfaces.- References.- 10 High Resolution Metal Replication of Freeze-Dried Specimens.- 1 Introduction.- 2 Electron Microscopy and Image Processing.- 3 Characterization of the Test Specimens.- 4 Controlled Freeze-Drying.- 5 High Resolution Shadowing.- References.- 11 Immunogold Labelling of Cryosections and Cryofractures.- 1 Introduction.- 2 Cryoultramicrotomy.- 3 Cryofractures.- 4 Label Efficiency.- 5 Conclusions.- References.- 12 Cryoultramicrotomy for Autoradiography and Enzyme Cytochemistry.- 1 Introduction.- 2 Cryoultramicrotomy for the Autoradiography of Diffusible Substances.- 3 Thin Cryosections in Histochemistry.- 4 Conclusions.- References.- 13 Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples.- 1 Introduction.- 2 Morphology.- 3 Analysis.- 4 Freezing.- 5 Instrumentation.- 6 Electron Interactions.- 7 X-Ray Emission.- 8 The Fracture Surface.- 9 Quantitative Analysis.- 10 Water Content or Dry Weight Fraction.- 11 Beam Damage and Mass Loss.- 12 Detection Limits.- 13 Conclusions.- References.- 14 Cryofixation of Dynamic Processes in Cells and Organelles.- 1 Introduction.- 2 Cooling Rate, Freezing Time, Time Resolution.- 3 Cellular Structures: Rapid Freezing Versus Chemical Fixation.- 4 Membrane Dynamics.- 5 Conclusions and Outlook.- References.- 15 Cryofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis.- 1 Introduction.- 2 Specimen Preparation.- 3 Some Biological Applications and Results.- 4 Conclusion and Outlook.- References.- IV Appendix.- 16 Safety Rules for Cryopreparation.- 1 Introduction.- 2 Asphyxiation with Gaseous Nitrogen.- 3 Gaseous Propane Explosions.- 4 Burns Caused by Secondary Cryogen Splashing.- 5 Burns Caused by Primary Cryogen Splashing.- 6 Ignition of Combustible Secondary Cryogens.- 7 Bursting of Cryogen Containers.- 8 Transport and Disposal of Cryogens.- 9 Concluding Remarks.- References.

Magnesium in newly formed dentin mineral of rat incisor.

Small amounts of magnesium are always detectable in addition to calcium and phosphorus in mineralized tissues such as dentin or bone. Magnesium has been considered to influence the mineralization process, especially crystal growth. The present study reports on the location and enrichment of magnesium in the newly mineralized dentin by using the high lateral resolution of energy dispersive X‐ray microanalysis combined with scanning transmission electron microscopy. To this end, we have used the continuously growing rat incisor as a model for a collagenous mineralizing system. Dental tissue was dissected free and cryofixed in liquid nitrogen–cooled propane. The distribution of elements was measured in freeze‐dried ultrathin cryosections. The magnesium distribution of the newly formed dentin area near the predentin area was found to be inhomogeneous. In certain small dentin areas, characteristical magnesium enrichments were observed. Further, high magnesium‐to‐phosphate molar ratios were found in these areas, and these were correlated with low calcium‐to‐phosphate molar ratios. Our results support the theory that magnesium is involved in the process of biological apatite crystal formation.

Cryofixation methods for ion localization in cells by electron probe microanalysis: a review

Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in‐situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion‐related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time‐controlled cryofixation is approximately 1 ms.

Two types of concretions in Drosophila Malpighian tubules as revealed by X-ray microanalysis: A study on urine formation

Abstract In the Malpighian tubules of Drosophila two types of luminal concretions are formed. Type-I concretions mainly accumulate high quantities of calcium and magnesium in a matrix of proteoglycans; type-II concretions accumulate potassium. Higher quantities of organic matrix are found in type-II concretions than in type I concretions. The latter are stored in the distal segment of the anterior Malpighian tubules while type-II concretions are formed between apical microvilli in the transitional and the middle segment of anterior tubules and in the distal and the middle segment of posterior tubules. In the tubules of the pupa and in the first meconium to be produced, the magnesium and calcium contents of excreted type-I concretions are similar. The outer envelope of the puparium exhibits only traces of calcium in contrast to the cuticle of the larva. Both types of concretions are products of excretion: they eliminate calcium and magnesium or potassium, respectively. During winter time and under normal photoperiods both types of concretions can join together to form large “biocrystals” inhibiting the flow of urine. Concretion formation is discussed as an economical method to excrete high amounts of cations in a condensed form with the loss of little water.

Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes

Cytotoxicity of the heavy metals gold, mercury, thallium and lead was studied by measuring the intracellular element distribution of cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections in a scanning transmission electron microscope. Exposure of the cells to aqueous solutions containing heavy metal ions in concentrations reaching a critical concentration caused increase of intracellular sodium and chloride content accompanied or followed by decrease of intracellular potassium content. Thus, the intracellular potassium/sodium ratio drastically decreased from control values of approximately 10 to values below 1 before changes of cell morphology became visible. In experiments with gold or mercury the decrease of the potassium/sodium ratio was preceded by transient cytoplasmic increase of sulfur and phosphorus. Heavy metal concentrations exceeding the critical concentration also caused an increase of cytoplasmic calcium concentration and finally decay of the cell structure. Cytotoxicity of heavy metals was found to increase in the order Pb, Au, Tl, Hg. Cytotoxic effects by Au, Tl or Hg in moderate concentrations were reduced by simultaneous addition of Zn or Pb to the culture medium. The results obtained prove electron probe X-ray microanalysis of cryosections as a sensitive probe of cell viability.

Potassium is Involved in Apatite Biomineralization

The biogenetic formation of mineral crystals, one aspect of biomineralization, is a multistep process of apatite formation throughout the growth of dentin tissue. An important step is the transformation of the non-mineralized predentin matrix to mineralizing dentin matrix and its biological control. In this study, the high capacity of elemental mapping is combined with single x-ray point measurements to elucidate whether special elements are involved in initiation or regulation of mineral nucleation. Directly at the mineralization front, micro-areas with a strong co-enrichment of phosphorus (e.g., as phosphate) and potassium are found. During the beginning of the calcium enrichment and the subsequent apatite mineral formation in the characteristic micro-areas, the content of potassium decreases significantly. These findings indicate that potassium is involved in the process of dentin mineralization.

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

SummaryCryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.

Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K+-dependent, amiloride-sensitive K+/H+ exchange system.

Tachyzoite calcium changes during cell invasion by Toxoplasma gondii

Abstract The invasion of host cells by the obligate intracellular protozoan parasite Toxoplasma gondii is calcium dependent. We have identified two calcium storage areas in tachyzoites, the endoplasmic reticulum and vesicles that contain high concentrations of calcium as amorphous calcium phosphate precipitates. Our data indicate that these vesicles slowly lose their calcium during the intracellular development of the tachyzoite as their nucleus phosphorus content increases. We found fluctuations in the sulfur content of the tachyzoite during invasion following the exocytosis of protein from the secretory organelles, with a loss of sodium and chlorine, and the uptake of potassium from the host cell cytoplasm. We demonstrated that penetration of the tachyzoite into the host cell was accompanied by increases in the concentrations of phosphorus and sulfur in the host cell nucleus, probably due to increased transcription. The cytosol sodium concentrations decreased, while the potassium content increased. Thus, the subcellular element distribution of tachyzoites and host cells changes during invasion and intracellular growth of the parasites. In addition, our results indicate that tachyzoite calcium might be involved in the egress of the parasite from the host cell.

Cryofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis

The interaction of the electron beam with the specimen not only provides information on its ultrastructure, but also on its elemental composition. In principle, the irradiating electron beam (probe) is focussed on the area of interest in the specimen, while the secondary radiation (e.g. X-rays) generated by the interaction with the specimen is processed through a spectrometer to provide information on the kind and amount of the elements present. Today, electron probe X-ray microanalysis (EPXMA) is the most widely used method in biological microanalysis. Also, electron energy loss spectrometry (EELS) acquires more and more attention. It would be beyond the scope of this book to explain the physical principles and the methodology of these analytical methods here, in particular, since there are numerous, well-written reviews on this subject (e.g. Chandler 1977; Hren et al. 1979; Hall and Gupta 1983; Morgan 1985). Rather, we want to discuss the particular importance of cryotechniques in this field and to point out the still existing problems of specimen preparation. Its crucial steps are not only relevant for EPXMA and EELS, but also for other microanalytical techniques such as proton probe X-ray microanalysis and laser probe mass spectrometry.