Dynamics of Golgi impregnation in neurons.

This paper describes the early stages of impregnation by the Golgi rapid method in sections and blocks of brain tissue. Aldehyde-fixed and potassium dichromate-treated sections of cerebral cortex were placed on glass slides and coverslipped. The dichromate solution was then replaced by a silver nitrate solution, and events taking place in the section were monitored and time-lapse recorded until the impregnation was interrupted and the sections subsequently prepared for electron microscopy. The tissue blocks, fixed and chromated in the same way, were placed into a silver nitrate solution for 30 min to 24 h and the progress of impregnation compared with the results obtained in the sections on the glass slides. Two basic modes of impregnation were observed, apparently in direct relation to the process of crystallization of silver chromate: crystals of silver chromate growing directly from the surface of the tissue into the nerve cell via its transected plasma membranes, and microcrystalline precipitate of silver chromate spreading into the nerve cell from nucleation centres dispersed in the tissue. The precipitate grows inside the cell as in a preformed channel until the cell has been filled. If the nucleation begins extracellularly, the precipitate extends into the narrow intercellular gaps. Electron microscopy showed that the crystalline precipitate consisted of multilamellar formations containing dense coalesced granules that did not cross plasma or endocellular membrane boundaries.     

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described.     

Identification of nerve fibres in the scanning electron microscope.

The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.     

An inexpensive spray-freezing unit for preparing specimens for freeze-etching

Photomicrographs of thick sections taken with reciprocal, lateral illumination in a compound microscope produce stereo-pairs. The method is particularly useful with thick celloidin sections of nervous tissue impregnated with the Golgi selective silver technique.     

The use of silver nitrate as a stain for scanning electron microscopy of arterial intima and paraffin sections of kidney

The suitability of silver nitrate as a stain for scanning electron microscopy was investigated. Accordingly en face preparations of arterial intima were impregnated with silver nitrate, fixed with formalin, and coated with platinum-palladium alloy. In SEM images, the silver lines surrounding the endothelial cells, and the deposits on the intima appear as white lines or dots on a darker background. Similar results were noted for nitrocellulose-embedded endothelium. Paraffin sections of kidney treated with silver nitrate (von Kossa's stain) were also examined. Deposits of the calcium salts of phosphates and carbonates (stained with silver nitrate) were easily differentiated from other tissue components. Results were compared with those obtained with the light microscope. The scanning electron microscopic examination provided a finer definition of the silver granules relative to the surrounding architecture. The limitations and advantages of these techniques and possible further applications of silver nitrate as a stain for scanning electron microscopy are discussed. The use of silver nitrate as a stain for some SEM preparations is recommended.     

Silver impregnation applied to striated muscle: The sarcoplasmic reticulum

The classical black reaction developed by Camillo Golgi is shown to impregnate the tubules and fenestrations of the sarcoplasmic reticulum (SR) in striated muscle. This is a double impregnation of chromate and silver, which usually fills extracellular spaces. The method is difficult insofar as long incubation times are required, and location of the successfully “stained” SR in plastic-embedded tissue blocks is unpredictable. The light microscope is absolutely necessary to find the good regions which can then be cut from the blocks in 1-μm-thick sections and examined in the electron microscope. Stereo pairs give the best results since these resolve overlap problems common to thick sections. A variety of artifacts are illustrated which can help avoid erroneous interpretations. The Golgi-“stained” SR shows this elusive network with unsurpassed contrast and should benefit the morphological studies of muscle-membrane enthusiasts.     

A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside-down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.     

Use of different morphological techniques to analyze the cellular composition of the adult zebrafish optic tectum

Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1 μm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.     

Quantitative aspects of synapses on Golgi-impregnated neurons.

With the classical Golgi techniques, numerous types of neurons can be distinguished in the cerebral cortex, each with a specific dendritic geometry and pattern of axonal ramifications. In the present review we describe two techniques which allow quantification of synapses on identified neurons: (1) Golgi-rapid impregnation-gold toning-electron microscopy, and (2) Golgi-Kopsch impregnation-gold toning-electron microscopy in combination with staining of the tissue with ethanolic phosphotungstic acid (E-PTA). Both techniques were applied on neurons in the visual cortex of young and adult rabbits. By means of rotating and tilting specimens in the electron microscope, the nondistinctive ultrastructure of obliquely sectioned synapses can be circumvented, leading to precise estimates of asymmetrical vs. symmetrical synapses without complete reconstruction of the neuron.     

Study of Golgi-impregnated material using the confocal tandem scanning reflected light microscope

The tandem scanning reflected-light microscope (TSM) is a real-time, direct-view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo-electric control of the objective lens was used to generate 3-D images from Golgi-impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35-mm colour film transparencies by photographing while automatically through-focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo-pairs were obtained by oblique through-focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a z map image as the focal depth at which the back-scattered light signal was maximal. The TSM was also modified so that a conventional transmitted-light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back-scattered-light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high-contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3-D arrangement of cell bodies and processes in the chosen volume.     

Application of the Golgi/electron microscopy technique for cell identification in immunocytochemical, retrograde labeling, and developmental studies of hippocampal neurons.

In this study the Golgi/electron microscopy (EM) technique has been used for an analysis of the fine structure, specific synaptic connections, and differentiation of neurons in the hippocampus and fascia dentata of rodents. In a first series of experiments the specific synaptic contacts formed between cholinergic terminals and identified hippocampal neurons were studied. By means of a variant of the section Golgi impregnation procedure, Vibratome sections immunostained for choline acetyltransferase, the acetylcholine-synthesizing enzyme, were Golgi-impregnated in order to identify the target neurons of cholinergic terminals in the hippocampus. It could be shown with this combined approach that cholinergic septohippocampal fibers form a variety of synapses with different target structures of the Golgi-impregnated and gold-toned hippocampal neurons. In this report cholinergic synapses on the heads of small spines, the necks of large complex spines, dendritic shafts, and cell bodies of identified dentate granule cells are described. The variety of cholinergic synapses suggests that cholinergic transmission in the fascia dentata is a complex event. Next, the Golgi/EM technique was applied to Vibratome sections that contained retrogradely labeled neurons in the hilar region of the fascia dentata following horseradish peroxidase (HRP) injection into the contralateral hippocampus. With this combined approach some of the hilar cells projecting to the contralateral side were identified as mossy cells by the presence of retrogradely transported HRP in thin sections through these Golgi-impregnated and gold-toned neurons. Our findings suggest that the mossy cells are part of the commissural/associational system terminating in the inner molecular layer of the fascia dentata. They are mainly driven by hilar collaterals of granule cell axons that form giant synapses on their dendrites. Finally, the Golgi/EM procedure was used to study the differentiation and developmental plasticity of hippocampal and dentate neurons in transplants and slice cultures of hippocampus. Under both experimental conditions, the differentiating neurons are deprived of their normal laminated afferent innervation but develop their major cell-specific characteristics including a large number of postsynaptic structures (spines). As revealed in thin sections of gold-toned identified cells, all these spines formed synapses with presynaptic boutons suggesting sprouting of the transplanted and cultured neurons, respectively. Altogether, the present report demonstrates the usefulness of the Golgi/EM technique, particularly of the section impregnation procedure, for a variety of studies requiring the identification of individual neurons at the ultrastructural level.     

Precipitation Procedures For Sodium,Potassium And Chloride Localization In Leaf Cells Of The Halophyte Suaeda Maritima

Attempts have been made to localize sodium and potassium with antimonate and cobaltinitrite reagents in Suaeda leaf segments by rapid aqueous and freeze-substitution precipitation procedures and to localize chloride by freeze-substitution precipitation with silver ions. Substantial sodium losses occurred from the tissue during both types of preparation although potassium and chloride losses were considerably reduced by the use of freeze-substitution when compared with the rapid aqueous procedures. The losses during freeze-substitution resulted from the required use of ethanol as substitution solvent (rather than acetone or diethyl ether, in which the reagents were much less soluble). Analytical electron microscopy showed that silver and cobaltinitrite specifically precipitated chloride and potassium respectively, but antimonate did not specifically precipitate sodium. The vacuoles were important sites of silver and antimonate deposits, although the chloroplasts were the major sites of cobaltinitrite localization. The value of these techniques in ion localization studies is discussed.     

Ultrastructural changes of the olfactory bulb in manganesetreated mice

The effect of manganese toxicity on the ultrastructure of the olfactory bulb was evaluated. Male albino mice were injected intraperitoneally with MnCl₂ (5 mg/Kg/day) five days per week during nine weeks. The control group received NaCl (0.9%). The olfactory bulbs of five mice from each group were processed for transmission electron microscopy after 2, 4, 6 and 9 weeks of manganese treatment. On week 2, some disorganization of the myelin sheaths was observed. After 4 weeks, degenerated neurons with dilated cisternae of rough endoplasmic reticulum and swollen mitochondria appeared. A certain degree of gliosis with a predominance of astrocytes with swollen mitochondria, disorganization of the endomembrane system, dilation of the perinuclear cisternae and irregularly shaped nuclei with abnormal chromatin distribution were observed after 6 weeks. Some glial cells showed disorganization of the Golgi apparatus. On week 9, an increase in the number of astrocytes, whose mitochondrial cristae were partially or totally erased, and a dilation of the rough endoplasmic reticulum were found. Neurons appear degenerated, with swollen mitochondria and a vacuolated, electron dense cytoplasm. These changes seem to indicate that the olfactory bulb is sensitive to the toxic effects of manganese.     

肉制品中氯化物含量测定——对氯化银沉淀排除方法的探讨

本文论述了应用佛尔哈德法测定肉制品中氯化物的含量,详细阐述了原理、试剂、步骤及其测定注意事项。主要阐述了氯化银沉淀对滴定终点判定的三种干扰排除方法：方法1、加入有机溶剂硝基汞;方法2、加入邻苯二甲酸二丁酯包裹氯化银沉淀;方法3、氯化银沉淀过滤,并通过实验数据比较这三种方法对实验结果的影响。结果表明三种方法均可行,但是有机溶剂毒性较强,氯化银沉淀过滤的方法实验过程较安全。考虑到准确、安全、经济三方面的因素,建议使用方法3。     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Electron microscopic study of the morphology and morphogenesis of virus of hemorrhagic fever with renal syndrome

The morphology and morphogenesis of the virus of hemorrhagic fever with renal syndrome (HFRS) and the associated ultrastructural changes in neurons of the infected mouse brain were examined by electron microscopy. The primary location of the infection in large neurons was in the Golgi apparatus, which had highly proliferated laminar and vesicular profiles. A small number of matured virus particles were found later individually or in small groups within the distended Golgi cisternae and vesicles. Most of the virus particles were round, oval, or elongated and measured about 70–110 nm in diameter. A lipid bilayered viral envelope with an external fringe of surface projections could be resolved at high magnification. The maturation (budding) of the virus occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Viral inclusion bodies containing fine filamentous material were seen frequently in close proximity to sites of virus maturation. The known morphological and morphogenetic characteristics of the virus particles observed in infected mouse brain gave further evidence for taxonomic identification of HFRS virus as a member of the family of Bunyaviridae.     

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.     

TEM characterization of Ge precipitates in an Al-1.6at% Ge alloy

The growth mechanism and morphology of Ge precipitates in an Al-Ge alloy was characterized by a combination of in-situ transmission electron microscopy, high-resolution transmission electron microscopy and three-dimensional electron tomography. Anisotropic growth of rod-shaped Ge precipitates was observed by in-situ transmission electron microscopy over different time periods, and faceting of the precipitates was clearly seen using high-resolution transmission electron microscopy and three-dimensional electron tomography. This anisotropic growth of rod-shaped Ge precipitates was enhanced by vacancy concentration as proposed previously, but also by surface diffusion as observed during the in-situ experiment. Furthermore, a variety of precipitate morphologies was identified by three-dimensional electron tomography.     

Electron cytochemical localization of cystine in plant cell walls

A method for specific, cytochemical staining of cystine residues was applied to thin sections of human hair keratin, two yeasts, one fungus and one green alga, for observations in the electron microscope. The method, which deposits granules of metallic silver at or close to the site of cystine residues, may be a useful means of investigating the detailed distribution of disulphide bonds in cells provided that the following criteria are rigorously applied. First, grains of metallic silver must remain after exhaustive sodium thiosulphate extraction; second, the deposition of grains of metallic silver must be inhibited by prior alkylation of the disulphide linkage. Finally, the method should be used in conjunction with other available means of analysis.