Localization of cations by pyroantimonate. I. Influence of fixation on distribution of calcium and sodium. An approach by analytical ion microscopy.

A modification of the potassium pyroantimonate (PA) method for localization of calcium and sodium was tested using skeletal muscle of mouse. Massive diffusion of these cations, depending on the method of fixation, was demonstrated by analytical ion microscopy (AIM) images on the optical microscopy level. Rapid penetration of the fixative appeared to be the principal condition that reduced diffusion of Ca2+ and Na+. Paraformaldehyde (2%) appeared more efficient than glutaraldehyde (1%) for preserving metal composition. Addition of 1% phenol strikingly improved the quality of the AIM images. Supersaturated PA (4%) appeared to retain about 10 times more sodium in the tissue than insaturated PA (2%). The role of different buffers is also discussed, particularly collidine, which permitted better preservation of sodium. Fixation with this buffer should be very useful for study by AIM of large-scale distribution of sodium. These results are analyzed at the ultrastructural level in the accompanying report.

Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin.

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

Rapid localization of carbon 14-labeled molecules in biological samples by ion mass microscopy.

We report here on the ability of secondary ion mass spectrometry (SIMS) to provide rapid imaging of the intracellular distribution of 14C-labeled molecules. The validity of this method, using mass discrimination of carbon 14 atoms, was assessed by imaging the distribution of two molecules of well-known metabolism, [14C]-thymidine and [14C]-uridine, incorporated by human fibroblasts in culture. As expected, 14C ion images showed the presence of [14C]-thymidine in the nucleus of dividing cells, whereas [14C]-uridine was present in the cytoplasm as well as the nucleus of all cells, with a large concentration in the nucleoli. The time required to obtain the distribution images with the SMI 300 microscope was less than 6 min, whereas microautoradiography, the classical method for mapping the tissue distribution of 14C-labeled molecules, usually requires exposure times of several months. Secondary ion mass spectrometry using in situ mass discrimination is proposed here as a very sensitive method which permits rapid imaging of the subcellular distribution of molecules labeled with carbon 14.

Inhaled soluble aerosols insolubilised by lysosomes of alveolar cells. Application to some toxic compounds; electron microprobe and ion microprobe studies.

Our previous investigations have shown that, after systemic injection, certain toxic aluminium, chromium, uranium, and cerium salts are reabsorbed and eliminated by the renal cells by an identical process in which lysosomes play a special role. The elements are precipitated in these organelles in the form of insoluble phosphate due to the activity of acid phosphatase. In this work we have studied the disposition of the same elements after aerosol inhalation of water-soluble particles. The microanalytic methods used, electronic probe and ionic microanalysis, enable the chemical composition of mineral intracellular inclusions and the distribution of elements in the tissues to be determined. The elements studied are precipitated in the lysosomes of alveolar cells (macrophages and type 1 pneumocytes) in the form of insoluble phosphate due to the action of acid phosphatase. These toxic elements do not cross the alveolar-capillary barrier and do not cause any lesion in the organism. Thus a new mechanism for the elimination of hydrosoluble particles has been demonstrated that is of great interest in the overall process of defensive reactions of the organism.

The distribution of indium in the central and peripheral nervous system of the young rat.

A subtoxic quantity of radioactive 111indium and stable indium was injected once into young rats. Subsequently pairs of these rats were killed at intervals of 15 minutes to 3 days after the injection. Tissues from the central (CNS) and peripheral nervous system (PNS) and blood were removed from each rat and the amount of 111indium was measured in the tissues. Cryostat sections of the cerebrum, cerebellum, spinal cord, sciatic nerve, a blood smear and a kidney control from each rat were examined with an analytical ion microscope (secondary ion mass spectrometry analysis) and the presence of 113 and 115 indium was localized in the tissues. These observations show that indium penetrates all neural tissues and that the brain resists the entry of indium - a very toxic element - much more effectively than the sciatic nerve.

Ion microscopy, a method for imaging the distribution of trace elements in the lung.

Analytical ion microscopy, a method of surface microanalysis, is applied to the detection of trace elements in the lung. With this method it is possible to obtain images of the distribution of any element in lung tissue sections with a resolution of 0.5 micron and with very high sensitivity (of an order of magnitude 3 or 4 times greater than with X-ray microanalysis). Under these conditions it is possible to study at the microscopic level the penetration into the lung cells of mineral materials administered in physiological amounts. The images are formed by ion microscopy of atoms sputtered as charged particles from the specimen, and these atoms are selected by mass spectrometry. This technique has been applied to the detection of aerosols (diam. less than 1 micron) of rare earths (thulium or cerium chloride), inhaled in very small amounts (20-50 micrograms), in cells from the lung of a rat. Images of the distribution of these elements were obtained in a few seconds or minutes, when X-ray microanalysis gave either a weak signal or none at all. Their behaviour in the lung could be easily studied several weeks after the end of the inhalation.

Detection by immunogold techniques of HIV antigens in Lowicryl ultrathin sections of infected cells.

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl K4M-embedded HIV-infected cells. With serum from an AIDS patient, specific immunogold labelling was obtained exclusively on mature viral extracellular structures. The more intense reactivity was obtained with core antigens. The present immunoelectron microscopy method provides several advantages - high sensitivity of immunodetection, good preservation of cellular morphology, easy preparation procedure - which could lead to the use of this method for HIV-infected human tissues.

An autoradiographic study of the in-vivo incorporation of [3H]-palmitic acid into the dentine and enamel lipids of rat incisors, with a comparison of rapid-freezing freeze-substitution fixation and aldehyde fixation.

Four and twenty-four hours after injection of the labelled precursor, there were twice as many silver grains scored with the cryotechniques as with conventional fixation. Absence of labelled-band formation in dentine, the diffusion of silver throughout the entire thickness of dentine as well as the layer of forming enamel. This suggests that lipids are membrane-associated components rather than true matrix-associated components.

Improved preservation of intramitochondrial granules in rat-incisor odontoblasts by rapid-freezing and freeze-substitution fixation.

Odontoblasts fixed by rapid-freezing and freeze-substitution, compared to cells fixed with aldehyde, exhibited a larger number of microfilaments, a granular material inside the rough endoplasmic reticulum, many electron-dense aggregates in cytoplasmic vesicles and numerous intramitochondrial granules. Although these granules were located in odontoblasts facing a well-mineralized layer of dentine, this did not support the possible existence of a transfer pathway for intracellular calcium which plays some part in mineralization processes.

A comparative fine structure study of rat cerebral cortex following ultra-rapid freezing and conventional chemical fixation procedures.

Adult rat (Sprague-Dawley) cerebral cortex was processed by ultra-rapid freezing at liquid helium temperature followed by freeze-substitution, osmium fixation and by other chemical, post-osmication procedures at 0-4 degrees and ambient temperatures as a comparative study for purposes of identifying differences and/or similarities in fine structure following these techniques. Five methods of processing were used: 1) rapid, slam-freezing at liquid helium temperature followed by osmium tetroxide/acetone freeze substitution; 2) perfusion with buffered, 2% glutaraldehyde at ambient temperature followed by post-osmication (2%); 3) en-bloc, buffered 2% glutaraldehyde fixation at 0-5 degrees centigrade and post-osmication (2%); 4) buffered, 2% osmium tetroxide perfusion at ambient temperature; and 5) en-bloc, buffered 2% osmium tetroxide fixation at 0-5 degrees centigrade. In ultra-rapid-frozen cortex good preservation was seen to a depth of 10-15 microns from the surface of the initial, copper-block contact. The tissue processed by ultra-rapid-freeze, freeze substitution demonstrates a general 'smoothness' of plasmalemmal and organelle membranes not observed in tissue prepared by chemical fixation alone. Cellular and organelle morphological differences were minor beyond the general 'smoothness' of membranes and a more intense background, electron density found in tissue prepared by rapid-freeze. Of particular interest was the practically identical images found in the four, chemical techniques not preceded by ultra-rapid freezing. High magnification images also revealed rather minor differences following ultra-rapid-freezing compared to tissue fixed by chemical fixation alone. Although these morphological differences are minimal, there can be no question of the fact that ultra-rapid-freeze followed by freeze-substitution is morphologically superior to chemical fixation alone. Ultra-rapid-freeze, eventually utilizing other substitution agents than osmium tetroxide, will offer several advantages and should be particularly useful for investigators involved in cytochemical and immunochemical methods.

[The detection of viral antigens and ribosomal RNA in cells by using low-temperature media]. / Vyiavlenie virusnykh antigenov i ribosomal'noi RNK v kletkakh s ispol'zovaniem nizkotemperaturnykh sred.

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl-embedded HIV-infected cells. A genomic probe containing ribosomal sequences and labeled with biotin was used to hybridize rRNA molecules in sections of animal cells embedded in Lowicryl. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression and viral proteins at the ultrastructural level.

[External adamantine epithelium, stellate reticulum and intermediate stratum of the enamel organ of the incisor in the rat: ultrastructural study of sections and after freeze fracturing]. / Epithélium adamantin externe, réticulum étoilé et stratum intermedium de l'organe de l'émail de l'incisive chez le rat: étude ultrastructurale sur coupes et aprés cryofracture.

A morphological study of the cells of the outer epithelium layer, of the stellate reticulum and of the stratum intermedium of the rat incisor on thin sections and after freeze-etching allowed the differentiation of an intracellular and an intercellular compartment. Both were implicated in transport and diffusion phenomenons. The structure of these cells suggested that they synthetize non exportable proteins. Nevertheless they could be implicated in some none precised activities of biosynthesis and secretion.

The distribution of filipin-cholesterol complexes in secretory ameloblasts of rat incisor.

The number of filipin-sterol complexes, visualised as 25-35 nm protuberances on freeze-fracture replicas of secretory ameloblasts of rat incisor, was small on the plasma membrane and nil on internal membranes of the cell body. Distal infoldings and vesicles inside Tomes process presented a higher number of protuberances. Such membrane-induced deformations were especially abundant on membrane remnants left inside the forming enamel and in holes at the dentine-enamel junction. This reflects regional variations of cholesterol or accessibility of cholesterol. Increased rigidity of membranous remnants may play a role in the induction of local defects during rat enamel formation. These observations clarify the origin of phospholipids and cholesterol detected biochemically.

Distribution of filipin-cholesterol complexes in rat incisor odontoblasts.

Using the polyene antibiotic filipin as a probe to visualize cholesterol on freeze-fracture replicas of membranes of young odontoblasts of rat incisor, we demonstrated that cholesterol-rich domains were present in cell processes, whereas only a small number of deformations induced by cholesterol-filipin interaction was observed in cell bodies. Except for a few large vesicles, intracellular membranes did not react with filipin. The large number of intramembrane particles observed on the plasma membrane of odontoblast processes did not affect the formation of visible filipin-cholesterol complexes.

Incorporation of (35S)sulfate and (3H)glucosamine into glycoaminoglycans in rat incisor predentine and dentine: comparison by autoradiography of fixation by rapid-freezing, freeze-substitution, and aldehyde fixation.

At 4 and 24 hours after injection of (35S)sulfate, there were 22-67% fewer silver grains on conventionally fixed sections than on sections fixed by cryotechniques. Differences were smaller when (3H) glucosamine was chosen as precursor. The number of silver grains increased between 4 and 24 hours in predentine and in a 30-micron band of dentine that had already undergone mineralization. Only a few grains were observed, however, in the 5-micron dentine band located at the mineralization front. This suggests that glycoaminoglycans, which in predentine have a space-filling role, facilitate transport and diffusion and inhibit mineralization, may limit crystal growth in dentine once a certain degree of apatite formation has been reached. All these properties are correlated with the structural and functional properties of the tissues.

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.

The appearance in TEM of proteoglycan predentine is fixation dependant.

The appearance of proteoglycans visualized on thin sections with alcian blue was compared in rat incisor predentine fixed either in aqueous aldehyde solutions or by anhydrous methods (acrolein vapours or rapid freezing-freeze substitution). The granular or branched chain-like structures detected after aqueous fixation probably resulted from precipitation or shrinkage of an expanded amorphous gel observed only after anhydrous fixation. In predentine, morphological interactions between proteoglycans and collagen fibres may be also artefactual.