Structural and functional alterations of cellular components as revealed by electron microscopy

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions. The study of intracellular vesicle compartmentalization is raising even more interest in the light of the importance of intracellular localization of mediators of the signaling in eliciting different biological responses. The study of the morphology of some intracellular organelles can supply information on the bio‐energetic status of the cells. TEM has also a pivotal role in the determination of different types of programmed cell death. In fact, the visualization of autophagosomes and autophagolysosomes is essential to determine the occurrence of autophagy (and also to discriminate micro‐autophagy from macro‐autophagy), while the presence of fragmented nuclei and surface blebbing is characteristic of apoptosis. SEM is particularly useful for the study of the morphological features of the cells and, therefore, can shed light, for instance, on cell–cell interactions. After a brief introduction on the basic principles of the main electron microscopy methods, the article describes some cell components with the aim to demonstrate the huge role of the ultrastructural analysis played in the knowledge of the relationship between function and structure of the biological objects. Microsc. Res. Tech., 76:1057–1069, 2013. © 2013 Wiley Periodicals, Inc.     

Are there gap junctions between chief (glomus,type I) cells in the carotid body chemoreceptor? A review

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.     

Evidence for ciliary pigment localization in colored ciliates and implications for their photosensory transduction chain: a confocal microscopy study

In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.     

Utilization of electron microscopic techniques in the in vitro study of adenohypophysial function and regulation.

Morphologic studies of human adenohypophysial cells using immunocytochemistry and electron microscopy have characterized the hormone-producing cell types of the normal gland and pituitary adenomas. The classifications which have emerged allow more accurate clinicopathologic correlations than ever before, but have also raised new questions concerning cytogenesis, pathogenesis, and structure-function correlations. We report the results of studies which marry the conventional morphologic techniques of light microscopy, immunohistochemistry, electron microscopy, and ultrastructural immunocytology with functional analyses using tissue culture and radioimmunoassay of hormones released into culture media. The hormone secretory activity of nontumorous and adenomatous pituitary cells is correlated with their structural features; their secretory responses to several adenohypophysiotropic factors are compared with morphologic alterations which are characterized at the light and electron microscopic levels by morphometric analysis. These studies have shown that hypothalamic stimulating hormones increase hormone release by their target cells and alter the ultrastructural appearance of the affected cells by increasing organelles involved in hormone synthesis. Inhibitory drugs and adrenal and gonadal steroids are capable of suppressing hormone release by some tumors and also give rise to morphologic changes which correlate with the functional inhibition. Hormone release by clinically nonfunctioning adenomas has been characterized and the behavior of these tumor cells in vitro sheds some light on the reasons for lack of clinical symptomatology. The plurihormonal nature of several nontumorous and adenomatous pituitary cell types has been characterized in vitro. The results of these studies provide the basis for more accurate structure-function correlations which can be used to study the hormonal milieu in vivo, to predict the role of pathogenetic factors in pituitary tumorigenesis, and to assess the therapeutic value of stimulating or inhibiting hormones and drugs.     

Effect of sodium azide on the ultrastructural preservation of tissues.

An electron microscopic study was carried out to examine the quality of ultrastructural preservation of parenchymatous and mesenchymatous tissues and isolated cells fixed in glutaraldehyde with sodium azide (NaN3) as an additive. The dense tissues fixed with conventional glutaraldehyde containing calcium chloride demonstrated only a narrow zone of good tissue preservation on the surface of the specimens. Addition of azide at a concentration of 0.1% greatly improved the cellular preservation in the deeper region of tissues, in particular with respect to the mitochondrial morphology. There was no adverse effect on other cell organelles. The improvement in mitochondrial preservation and the enhancement of penetration of the fixative is presumably due to selective and instantaneous inhibition of mitochondrial metabolic activity by the azide, thus retarding anoxic degenerative effects on cellular structures until permanent fixation is completed by the comparatively slow-acting aldehyde. However, the addition of azide offers no significant improvement in the ultrastructural preservation of isolated lymphocytes and liver cells, or fibroblasts maintained in culture.     

Current basis for the ultrastructural clinical diagnosis of tumors: A review

The ultrastructural diagnosis of tumors requires a careful analysis that should be done in an orderly fashion. It requires precise planning from the time of specimen collection to the selection of the area to be examined. Pictures must be taken systematically and every micrograph should allow to answer whether the number of cells photographed is adequate; whether mitoses are present, what is the pattern of the tumor; what is the appearance of the cell membrane; whether the cells are joined by junctional complexes; whether free surfaces possess microvilli or cilia; what organelles are present and how they are distributed; whether there are secretory granules, melanosomes, or other cytoplasmic elements. Nuclear and nucleolar size and shape have to be taken into consideration. The composition of the interstitial extracellular matrix is important in certain types of tumors. Although these questions are not the only ones to be addressed, their use in a logical fashion is helpful when it concerns the ultrastructural diagnosis.     

High-Resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.     

Peripheral sensilla of some lower invertebrates: the Platyhelminthes and Nematoda.

The flatworms (Platyhelminthes) and the round worms (Nematoda) are phyla exhibiting strikingly different levels of cellular organization. In both, sensilla are composed of the endings of sensory dendrites intercalated into their epidermis. In flatworms, sensilla that penetrate the syncytial epidermis bear sensory processes derived from cilia. In free-living species, the sensory processes more closely resemble motile cilia, while in parasites, greater deviations occur from the classical cilium pattern. Estimates of the function of the various sensilla have been largely arbitrary, and remain based on ultrastructural features. Sensilla in round worms lie below or within a heavy secreted cuticle. Two glia-like cell types occur. The socket cell mediates contact with cuticle and is responsible for cuticular modifications essential for operation of the sensillum. The sheath cell forms a receptor cavity around the sensory processes and regulates its environment. Sensory processes vary greatly from the classical cilium pattern. Absence of a basal body, but preservation of a ciliary necklace, suggests that the latter has a primary importance in sensory transduction. Estimates of function are based largely on ultrastructural features and analogies to arthropod sensilla. Genetic studies with the free-living nematode Caenorhabditis are beginning to demonstrate details of function and development. Speculations on the roles of basal bodies, rootlets, and vesicles and on the significance of recessed sensilla are given.     

A fluorescence scanning electron microscope

Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.     

Ultrastructural studies define soluble macromolecular,particulate, and cellular transendothelial cell pathways in venules,lymphatic vessels,and tumor-associated microvessels in man and animals

We present de novo studies and review published efforts from our laboratory, spanning 12 years (from 1988 to 2000), where we have used ultrastructural approaches to study the functional anatomy of the microvasculature in man and animals in health and disease. These efforts have defined a new endothelial cell organelle, termed the vesiculo-vacuolar organelle (VVO), which participates in the regulated transendothelial cell passage of soluble macromolecules. The studies defining this organelle utilized ultrathin serial sections, three-dimensional computer-assisted reconstructions, and ultrastructural electron-dense tracers to establish luminal to abluminal transendothelial cell continuity of VVOs. Commonality of VVOs and caveolae is suggested by the ultrastructural anatomy of individual units of VVOs and caveolae, the presence of caveolin in both structures, and a mathematical analysis of morphometric data, all of which suggest that VVOs form from fusions of individual size units equivalent to vesicles of caveolar size. Ultrastructural studies have localized potent permeability factors and their specific receptors to VVOs in in vivo tumor and allergic inflammation models. Regulation of permeability through VVOs has been quantified and shown to be increased in tumor microvessels and in control vessels exposed to potent permeability-inducing mediators. The transendothelial cell passage of particulate macromolecules occurs by vacuolar transport in tumor vessels; in permeability factor-exposed control vessels, colloidal carbon traversed endothelial cells via the development of pores that did not communicate with or disrupt intercellular junctions by gap formation. Serial section and computer-assisted reconstructions established these findings and suggested the possible development of transendothelial cell pores from VVOs. Serial sectioning and computer-assisted three-dimensional reconstructions of ultrastructural samples of an acute inflammation model revealed a transendothelial cell traffic route for motile neutrophils and platelets in the absence of classical ultrastructural criteria for regulated secretion from either cell.     

Review: Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.     

Physiology and pathophysiology of the human spermatozoon: The role of electron microscopy

In this article, the major contributions of electron microscopy to the present understanding of the physiology and pathophysiology of the human spermatozoon are reviewed. The ultrastructural organization of sperm organelles playing a significant role for cell function and, therefore, for the reproductive process is described. Also, the major abnormalities and defects of the various organellar systems and how they impair the reproductive function and/or the viability of the cell are reviewed.     

Uranyl sulphate: A new negative stain for electron microscopy

Uranyl sulphate is a negative stain of high quality for electron microscopy of macromolecules below their isoelectric point. The stain results in good contrast and high resolution as demonstrated by optical diffraction of periodic structure. Analysis of scanning transmission electron microscopic data reveals that uranyl sulphate is lower in background noise level than uranyl acetate and is dramatically more resistant toward granularization upon continued exposure to electron irradiation. Electron microscopic images of most macromolecules contrasted with uranyl sulphate were indistinguishable from those obtained with uranyl acetate. However, electron microscopic images of Reo virus contrasted with uranyl sulphate are always readily distinguished from those obtained with uranyl acetate.     

Intracellular distribution of actin in cells of the orgen of Corti: A structural basis for cell shape and motility

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post-embedding staining of Lowicryl K4M sections, pre-embedding staining of permeabilized cells of the organ of Corti, pre-embedding staining of vibratome sections, and pre-embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent—conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.     

Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy.     

Analysis of directly frozen macromolecules and tissues in the field-emission STEM

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (? 10³ e/nm²) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm². For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm² regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.     

Ultrastructural aluminium detection in amphibian tissues by electron spectroscopic imaging and electron energy-loss spectroscopy

Aluminium causes a variety of toxic effects in living organisms but very little is known about its uptake, pathways and locations of deposition. We have applied electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) to locate aluminium at the ultrastructural level in amphibian larvae from acidic ponds. It is found diffusely bound or precipitated in cell organelles. The spatial resolution of aluminium detection is high. The elemental composition of small areas can be demonstrated by EELS. Three different fixation procedures give similar results. The two- and three-window methods at the K- and L-edges are compared.     

Isolation and preparation of colonies grown on soft agar for ultrastructural investigation

Preparation of cell colonies grown on soft agar for electron microscopic examination is tedious. Mechanical damage to cells can occur during the process of colony removal. A procedure which permits rapid colony removal and preserves the integrity of the individual colony for ultrastructural examinations is described in this paper.     

Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 X, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.