Osmium Postfixation Research Articles

Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation

BackgroundDense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. New MethodHere, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 μm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. ResultsMultiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Comparison with Existing MethodsVolumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT’s advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. ConclusionsDeliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.

Atlas of Normal Microanatomy, Procedural and Processing Artifacts, Common Background Findings, and Neurotoxic Lesions in the Peripheral Nervous System of Laboratory Animals.

The ability to differentiate among normal structures, procedural and processing artifacts, spontaneous background changes, and test article-related effects in the peripheral nervous system (PNS) is essential for interpreting microscopic features of ganglia and nerves evaluated in animal species commonly used in toxicity studies evaluating regulated products and chemicals. This atlas provides images of findings that may be encountered in ganglia and nerves of animal species commonly used in product discovery and development. Most atlas images are of tissues from control animals that were processed using routine methods (ie, immersion fixation in neutral-buffered 10% formalin, embedding in paraffin, sectioning at 5 µm, and staining with hematoxylin and eosin) since these preparations are traditionally applied to study materials produced during most animal toxicity studies. A few images are of tissues processed using special procedures (ie, immersion or perfusion fixation using methanol-free 4% formaldehyde, postfixation in glutaraldehyde and osmium, embedding in hard plastic resin, sectioning at 1 µm, and staining with toluidine blue), since these preparations promote better stabilization of lipids and thus optimal resolution of myelin sheaths. Together, this compilation provides a useful resource for discriminating among normal structures, procedure- and processing-related artifacts, incidental background changes, and treatment-induced findings that may be seen in PNS tissues of laboratory animals.

Adapted methods for scanning electron microscopy (SEM) in assessment of human sperm morphology.

Infertility is a widespread problem, and in some cases, the routine basic semen analysis is not sufficient to detect the cause of male infertility. The use of the scanning electron microscope (SEM) could provide a detailed insight into spermatozoa morphology, but it requires specific sample preparation techniques. The purpose of this study was to select, adjust, and optimize a method for the preparation of spermatozoa samples prior to SEM analysis, and to establish the protocol required for its use in clinical practice. We examined sperm samples of 50 men. The samples were fixed with modified iso-osmolar aldehyde solution followed by osmium post-fixation. In the first method, dehydration of the cells and subsequent critical point drying (CPD) were performed on a coverslip. In the second method, the samples were dehydrated in centrifuge tubes; hexamethyldisilazane (HMDS) was used as a drying agent instead of CPD, and the samples were air-dried. The third procedure was based on a membrane filter. The samples were dehydrated and dried with HMDS in a Gooch crucible, continuously, without centrifugation or redispersion of the sample. Our results showed that the fixation with modified iso-osmolar aldehyde solution followed by osmium post-fixation, and combined with dehydration and CPD on a coverslip, is the most convenient procedure for SEM sample preparation. In the case of small-size samples or low sperm concentration, dehydration and drying with HMDS on the membrane filter enabled the best reliability, repeatability, and comparability of the results. The presented procedures are suitable for routine use, and they can be applied to confirm as well as to correct a diagnosis.

A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryzias latipes) eggs and eleutheroembryos as lipid rich models

Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi-ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and gamma-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.

Immunocytochemistry of renal membrane proteins on epoxy sections

Immunocytochemistry performed on paraffin or cryosections is often hampered by poor morphology. Epoxy sections, in contrast, generally retain well-preserved tissue architecture. Immunocytochemistry, however, on epoxy-embedded sections is difficult due in part to the plastic itself and to the fixation conditions. Here, we present a technique for visualization of membrane proteins by immunocytochemistry on epoxy sections of kidneys fixed with glutaraldehyde without or with osmium post-fixation. Semithin sections were obtained from Epon 812-embedded mouse and rat kidney blocks. Before immunoperoxidase or immunofluorescence labeling, the sections were etched with the epoxy solvent, methanolic potassium hydroxide, followed by antigen retrieval using microwave heating. The sections were then treated with the primary antibody followed by secondary antibodies as usual. The distribution and expression patterns of a variety of membrane proteins, such as aquaporin (AQP)-1, AQP-2, and megalin, were identical to those observed by traditional immunocytochemical procedures on paraffin or cryosections. The advantages of our novel method include not only enhanced morphological quality but also the feasibility for investigators to visualize antigens of interest using archival specimens in Epon blocks.

The Transillumination Possibility of Imidazole–Osmium Postfixed Tissue and Its Consequences for the Handling of Tissue Samples

Osmium postfixation is established as a routine procedure for transmission electron microscopy (TEM). On the one hand, this routine procedure leads to good results for TEM, but on the other hand results in blackened tissue samples that do not allow examination of any structures within the embedded tissue sample by a light microscope. Equivalent fixation results for TEM are achieved with imidazole-osmium postfixation, and with this postfixation method tissue is not blackened and can be transilluminated with point light sources. This allows easier recognition of histological details within tissue samples and makes it possible to screen embedded samples for appropriate ultrastructural processing. Jejunum is used to demonstrate the method.

The transillumination possibility of imidazole-osmium postfixed muscle tissue and its consequences for the handling of muscle tissue samples.

The osmium postfixation of tissue leads to good results for transmission electron microscopy, but also produces completely blackened tissue samples that do not allow the recognition of internal structures. With imidazole-osmium postfixation, one achieves comparable results in high electron microscopic resolution as with routine osmium postfixation. But the tissue samples are not blackened and can, therefore, be transilluminated with point light sources. The new postfixation technique makes it possible to recognize histological details such as vessels, nerve fibers, and the cross-banding pattern in an untrimmed block. This makes it possible to screen-embedded tissue samples for appropriate ultrastructural processing.

Malachite green and phthalocyanine-silver reactions reveal acidic phospholipid involvement in calcification of porcine aortic valves in rat subdermal model.

Subdermal implant models are helpful in the study of calcification "in vivo" and for testing anticalcific treatments. After implantation of porcine aortic valve leaflets in rat subcutis, we previously found that glutaraldehyde-Cuprolinic blue reactions (GA-CB) at low pH induce favourable tissue unmasking from mineral deposits, and visualize peculiar, electrondense layers that outline the calcifying cells and matrix vesicle-like structures. The layer-forming material seemed to consist of acidic phospholipids because of its anionic nature and differential susceptibility to chemical/enzymatic extractivity. In the present investigation, pre-embedding glutaraldehyde-Malachite green (GA-MG) reactions and subsequent osmium post-fixation were compared with pre-embedding GA-CB reactions, combined with post-embedding von Kossa silver staining (GA-CB-S), to assess whether the layer-forming material is actually composed of acidic phospholipids and exhibits calcium-binding properties. After lowering standard pH, GA-MG reactions also caused sample demineralization and the appearance of pericellular osmium-MG-reactive layers comparable to CB-reactive ones. Moreover, GA-CB-S reactions showed that major silver precipitation was superimposed to the CB-reactive layers, whereas minor metal extra-precipitation occurred at three distinct, additional sites. These results demonstrate that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface, replacing cell membrane and acting as major apatite nucleator. However, the overall observations are consistent with the hypothesis that certain phases are common to the various types of normal and/or abnormal calcification.

Transmission electron microscopy of yeast

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum. Microsc. Res. Tech. 51:496–510, 2000. © 2000 Wiley-Liss, Inc.

Transmission electron microscopy of yeast

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum.

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone.

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16-18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4M guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an M(r) of approximately 73000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation.

Ultrastructure of Verdigellas peltata (Palmellaceae, Chlorophyta), a deep-water, palmelloid alga with ferritin and trilaminar sheaths

The palmelloid green alga Verdigellas peltata D.L. Ballantine et J.N. Norris was collected from depths of 70 to 110 meters in the Bahamas and studied by light microscopy and transmission electron microscopy. Most of the cell was filled by a cup-shaped chloroplast that contained scattered starch grains but no pyrenoid. Some chloroplasts had clusters of granular inclusions that were identified as ferritin based on their particle size, native electron density, and iron content; the last was detected by energy-dispersive X-ray microanalysis. A nucleus, dictyosome, mitochondrion, and large vacuole occupied a pocket mostly surrounded by the chloroplast. Centrioles and basal bodies were absent. The nuclear envelope was continuous with the endoplasmic reticulum, and some cells had one or two spherical, glycoprotein inclusions, c. 1–2 μm in diameter, within the confluent lumen. The Golgi vesicles contributed fibrillar material to the vacuole, but most of the vacuolar contents consisted of appressed sheets of trilaminar material. Trilaminar sheets were also present between the plasmalemma and cell wall. Preparation of specimens without osmium postfixation resulted in the loss of membrane contrast, but the trilaminar sheets were unaffected. The cell walls apparently thicken by the addition of trilaminar sheets to their inner surface. The part of the thin cell wall overlying the cytoplasmic pocket was often perforated by pores. The palmelloid colonies contained an abundance of acetolysis-resistant material.

Myelination of the ventral and dorsal roots of the C8 and L4 segments of the spinal cord at different stages of development in the gray opossum,Monodelphis domestica

We have quantified the number and size of myelinated fibers of the ventral and dorsal roots of selected segments that innervate the forelimbs (C8) and hindlimbs (L4) in the developing opossum, Monodelphis domestica. The gray opossum was chosen because it is born very immature and its somatomotor development occurs almost entirely postnatally. After aldehyde fixation, osmium postfixation, and resin embedding, the roots were cut transversely (1.5 pm), stained with toluidine blue, and observed and photographed by using light microscopy. The counts and measurements were made with a digitizing table. Myelination of the C8 and L4 roots begins during the second week of life and occurs according to two gradients: rostrocaudal and ventrodorsal. The number of myelinated fibers in these roots increased over approximately 7 weeks after which an excess, compared with their adult value, was recorded during the following weeks in three of the four roots. The supernumerary myelinated fibers are presumed to be collaterals. The fibers increased in diameter until at least 98 days. The classification according to size for the ventral roots (alpha and gamma) became evident in the fourth week, but that in types I, II, and III for the dorsal roots was never clear. There was no significant difference in the number and size distribution of myelinated fibers between sexes until late in development. The fibers innervating the limbs thus become myelinated postnatally in the opossum, a process that occurs over a protracted period and that continues after sensorimotor reflexes and locomotion appear adult-like.

Light and Electron Microscope Immunohistochemical and Biochemical Studies of Vitronectin in Developing Rat Bone.

Recently, Seiffert using light microscope (LM) immunohistochemical methods, has localized vitronectin (Vn) in the mineralized bone matrix of mature mouse long bone after demineralization with 10 % nitric acid, indicating that Vn is a specific component of bone tissue. This raises the possibility that Vn is involved in regulation of bone metabolism. The present study extends previous studies to the electron microscope (EM) level and utilizes biochemical methods to determine the distribution and nature of Vn in early bone formation of developing rat mandible with rabbit antimurine Vn IgG (antiVn).Developing jaws of fetuses were collected at embryonic day 15-18 from pregnant Wistar rats. After al-dehyde fixation, specimens with and without osmium post-fixation were dehydrated, and embedded in paraffin, Spurr's resin, or LR gold resin for morphological and immunohistochemical observations. Sections cut from paraffin- or LR gold resin-embedded specimens were immunostained with antiVn, which was kindly provided by Dr. D. Seiffert, The Scripps Research Institute, La Jolla, California.

Light and Electron Microscope Immunohistochemical Localization of Decorin and Biglycan in Osteoblasts and Early Bone Matrix of Developing Rat Bone.

Decorin and biglycan are the predominant proteoglycans (PGs) isolated from bone of several animal species. Previous light microscope (LM) immunohistochemical studies of fetal human bone demonstrated that osteoid and osteoblasts reacted with both biglycan and decorin antibodies, but min-eralized matrix did not; however, the precise distribution of immunostaining was not examined at the electron microscope (EM) level. The present study examines LM and EM immunolocalization of decorin and biglycan in osteoblasts and early bone matrix of developing mandible of fetal rats, using polyclonal antibodies (LF113, LF106) directed against synthetic peptides corresponding to nonho-mologous regions of the two core proteins.Fetuses were collected at embryonic day 15-18 from pregnant Wistar rats. After aldehyde fixation, developing jaws with and without osmium post-fixation were dehydrated, and embedded in paraffin, Spurr's resin, or LR gold resin for morphological and immunohistochemical observations. Sections cut from paraffin- or LR gold resin-embedded specimens were immunostained with LF113 specific for rat decorin or LF106 specific for rat biglycan, which were kindly provided by Dr. L.W. Fisher, National Institute of Dental Research, NIH, Maryland.

Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus.

To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.

Use of Nanogold Followed by Silver Enhancement and Gold Toning for Preembedding Immunolocalization in Osmium-fixed, Epon-embedded Tissues

A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and embedding in Epon for good structural visualization. The technique was applied to rat testis with anti-laminin and anti-fibronectin antibodies, showing that these two proteins are localized differentially in the lamina propria of the tissue.

Liver of juvenile Atlantic salmon, Salmo salar L.: a light, transmission, and scanning electron microscopic study, with special reference to the sinusoid.

This report provides a detailed description of sinusoidal and perisinusoidal structures in the normal liver of the juvenile Atlantic salmon (Salmo salar L.), a teleost species. The liver was studied by light, transmission, and scanning electron microscopy, and organ specimens were sampled after retrograde, whole-body perfusion through the dorsal aorta using 3% glutaraldehyde. Detailed characterization of perisinusoidal stellate cells also included immunohistochemical staining for desmin and evaluation of autofluorescence of the same cells upon excitation in ultraviolet (UV) light. The sinusoid is lined by one cell type only: the endothelial cell. No intraluminal pit cells or Kupffer cells are present. The space of Disse contains reticulin fibres, visualized by Gomori's silver stain, and perisinusoidal stellate cells (PSC). PSC exhibited autofluorescence in UV light, indicating that these cells store vitamin A in cytoplasmic lipid droplets. Immunohistochemically, PSC were found negative for desmin. The space of Disse, extending deep down between adjacent hepatocytes, receives long, slender microvilli from parenchymal cells. In addition to scattered macrophages, interhepatocytic cells (IHC) are found perisinusoidally. Hepatocytes of Atlantic salmon form branching and anastomosing tubules. The sinusoids of Atlantic salmon liver are lined by a fenestrated endothelium, with PSC located in the space of Disse, with macrophages and IHC as inhabitants of the interhepatocytic space. IHC show ultrastructural similarities to mammalian pit cells and teleostean large granular lymphocytes, as well as to piscine monocytes. PSC might be storage cells for vitamin A in Atlantic salmon as shown by autofluorescence in these cells, while immunohistochemical studies indicate that desmin does not seem to be an adequate immunohistochemical marker for PSC in the juvenile Atlantic salmon. Methodologically, fixation for electron microscopy was performed by a new and convenient perfusion method: arterial retrograde whole body perfusion. Liver specimens intended for scanning electron microscopy were fractured at room temperature after prolonged osmium postfixation, leaving hepatocytes intact and producing images well suited to document the three-dimensional structure of cells and tissue.

Modulation of the Golgi apparatus in Saccharomyces cerevisiae sec7 mutants as seen by three‐dimensional electron microscopy

The three-dimensional configuration of the Golgi apparatus has been examined with the electron microscope in thick Golgi sections of Saccharomyces cerevisiae prepared from a wild-type strain and from sec7 mutants maintained for various periods of time at the nonpermissive temperature of 37 degrees C and then returned to the permissive temperature of 24 degrees C. Reduced osmium postfixation of glutaraldehyde fixed specimens stained intensely the content of Golgi elements and thus facilitated their three-dimensional characterization. In wild-type S. cerevisiae, the Golgi elements usually appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. Along such networks, distensions filled with stained material were similar in size to nearby secretory granules, suggesting that the latter formed by fragmentation of the Golgi elements. In sec7 mutants maintained at 37 degrees C in low (0.1%) glucose medium, secretion granules progressively decreased in number and soon disappeared. Concomitantly the networks of Golgi tubules increased in size and complexity, lost their distensions, and then transformed into flattened saccules forming stacks of up to seven or eight saccules that were similar to the Golgi stacks seen in mammalian cells. However in contrast to the latter, connections between the saccules were evident and Golgi-associated small vesicles were generally absent. Following return to the permissive temperature (24 degrees C), secretion granules reappeared, the Golgi stacks progressively decreased in size, and resumed their initial state of separated small tubular networks. Thus in sec7 mutant, grown at 37 degrees C in low glucose medium, segregation of secretory granules is blocked. As a result, Golgi membranes accumulate to form a continuous system of stacked and interconnected saccules.