Electron microscopy of body fluids

Transmission electron microscopy and scanning electron microscopy are complementary techniques; the former is useful for examining the fine internal structural detail of tissue and cell surfaces and the latter is useful for studying three-dimensional configurations. The methods and findings of transmission and scanning electron microscopic study of effusions are summarized, and their practical application in routine clinical work is assessed.     

Electron microscopy of leukemias and lymphomas

Modern advances in the diagnosis and treatment of the leukemias and lymphomas make use of dynamic techniques in immunology, genetics, and molecular biology, and, as a result, morphology represents a smaller proportion of the total diagnostic work-up of these diseases today than heretofore. However, morphology still remains a basic and necessary starting point in the study of these diseases, as is evidenced in the continuing general application of classifications such as the FAB (French-American-British) for the leukemias, and the "Working Formulation" (Working Formulation of Non-Hodgkins Lymphoma for Clinical Usage) for the lymphomas. Although accurate interpretation of cell-type is often possible at the light microscopic level, there are other occasions when electron microscopy may be necessary or useful. Furthermore, a familiarity with the ultrastructure of hematopoietic, lymphoid, and other cells should increase the pathologist's and hematologist's diagnostic acumen and, perhaps, even enhance his or her perspective at the light microscope level.     

Electron microscopy of serum sickness nephritis

The renal lesions of serum sickness were studied with the electron microscope. The most prominent change was a marked swelling and proliferation of glomernlar endothelial cells causing obliteration of the capillary lumen. The basement membrane also showed focal thickenings and excrescences. Deposits of electron-dense material blended into the basement membrane. On the extracapillary side epithelial foot processes were reduced in number and replaced by broad sheets of cytoplasm which were closely applied to the basement membrane. From a comparison of electron and fluorescent microscopic studies of the glomerulus in serum sickness, it would seem that antigen-antibody complexes initiated injury in endothelial cells, although the possibility of the primary reaction occurring on basement membrane cannot be excluded.     

Electron microscopy of salivary gland viruses

A human and a mouse strain of the salivary gland virus have been examined by electron microscopy. The human strain was transmitted, prior to examination, to tissue cultures derived from human myometrial cells, while the mouse strain was examined in mice inoculated intraperitoneally. The nuclear forms associated with both strains of virus were morphologically similar. Nuclear inclusions, composed of particles interspersed with dense clumped chromatin, were a striking feature of infected cells. The cytoplasmic forms were of 2 types—one a 300 to 500 mµ homogeneous dense spherical form, and the other a target-like form composed of a central dense dot in a pale zone surrounded by a dense shell—the entire configuration measuring 100 to 180 mµ. The target-like particle appeared to be identical in both strains. The spherical cytoplasmic forms in cells infected with the human strain appeared to be solid, while in cells infected with the mouse strain there was evidence of formation of target-like forms within the spheres. Possible mechanisms by which infection of the cell may occur, as well as possible mechanisms and sites of multiplication of virus, are discussed.     

Electron microscopy of chronic eosinophilic pneumonia

We have investigated two cases of chronic eosinophilic pneumonia using the electron microscope. The alveolar septa were thickened due to edema and an infiltrate of numerous mononuclear cells and eosinophils, with a few lymphocytes and occasional plasma cells. Macrophages were often located close to eosinophils and extracellular eosinophilic granules. Occasional eosinophilic granules were observed in the cytoplasm of mononuclear cells. The most striking finding was the presence of distinctive elongated, narrow, tubular inclusions in the cytoplasm of several of the mononuclear cells. These inclusions presented complex curved profiles which sometimes terminated in small, dilated, dense vesicles. Some of the narrow tubular sections of the inclusions presented a pentalaminar structure. Elsewhere, the tubular structures showed localized globular dilatations which contained granular material. Elongated strands of electron-dense material, identical to that forming the intracytoplasmic inclusions, were also located extracellularly, between adjacent mononuclear cells and between mononuclear cells and eosinophils. These inclusions are considered to be the product of phagocytosis of cellular debris and to be related to phagolysosomes rather than to Birbeck granules.     

Electron microscopy of isolated human hepatocytes

Summary We have investigated, by scanning and transmission electron microscopy (SEM and TEM), the cell surface morphology of isolated human hepatocytes. For this purpose, liver cells were mechanically isolated from surgical or needle liver biopsies, fixed in 3% glutaraldehyde and post-fixed in 2% osmium tetroxide. In order to handle a low number of cells, a particular procedure for harvesting hepatocytes on coverslips has been developed for SEM and anin situ embedding procedure in polyethylene-embedding capsules was applied for TEM. A rough membrane exhibiting short, uniform microvilli and pores of 0.1 μ in diameter was the main feature of isolated liver cells. Furthermore, single hepatocytes showed no polarity and junctional or bile canaliculus remnants were rarely observed. However, differences in surface configuration were noted in relation to culture conditions, such as oxygen and temperature during isolation procedures. SEM, when controlled by TEM for intracellular preservation, is proposed as a reliable method for screening small quantities of hepatocyte suspensions, for intact cells and for the study of surface configuration under experimental conditions. Part of this work was made possible by a grant from the Swiss National Science Foundation (grant no. 3.849.081), Dr. A. Trevisan is a recipient of a grant fromConsiglio Nazionale delle Ricerche (CNR), Roma, Italy, for research on viral hepatitis.     

Electron microscopy of HeLa cells infected with adenoviruses

HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies.     

Electron microscopy in determining origin of metastatic adenocarcinomas

Light microscopy has limited value in predicting where the primary site of a metastatic adenocarcinoma might be located. In our series, electron microscopy was useful in determining the primary site in approximately 85% of patients with a metastatic adenocarcinoma of unknown origin. This study is based on the ultrastructural examination of more than 100 such cases. In the remaining 15% of the cases, electron microscopy usually provided assistance in reducing the differential diagnosis to a minimum, usually to two possible primary sites. The majority of the metastatic adenocarcinomas showed rather specific ultrastructural features to suggest the site of origin. This application of electron microscopy has never been fully explored and has considerable clinical importance and economic impact in health care systems. An extensive work-up to determine the primary site, with its inconvenience to patient and family, as well as delay in adequate treatment, can be avoided with early diagnosis of the primary site.     

Electron microscopy of Pseudomonas aeruginosa treated with sulbenicillin and dibekacin

A possible mechanism responsible for the combined effects of sulbenicillin and dibekacin on Pseudomonas aeruginosa IAM 1007 was investigated. The bactericidal activity of the above two drugs in combination was very strong. The regrowth of test strains after removal of the drugs was suppressed markedly, even when they were exposed to sulbenicillin plus dibekacin at a subinhibitory concentration of individual drugs. Sulbenicillin caused elongation of the bacterial cells. At the early stage of elongation, no demonstrable changes of ultrastructure of the cell wall were observed. At the late stage, lysis of the peptidoglycan layer occurred and spheroplast was formed. However, most of the outer membrane of the cell wall remained intact. Sulbenicillin acts upon the peptidoglycan layer, but not on the outer membrane. Thus it is difficult for sulbenicillin alone to cause cell lysis. On the other hand, dibekacin caused destruction of ribosomes and lysis of the outer membrane of the cell wall. Both sulbenicillin and dibekacin act on the cell wall, the former on the peptidoglycan layer (the inner membrane) and the latter on the outer membrane. The combined use of sulbenicillin and dibekacin caused elongation of bacilli and severe destruction of the inner and outer membranes of the cell wall. These morphological changes occurred even when the concentration of the individual drug was lower than its minimum inhibitory concentration (MIC). Furthermore, the cells elongated by sulbenicillin were ruptured easily when treated with dibekacin subsequently. The bacilli treated with dibekacin at a concentration lower than MIC and then treated with sulbenicillin at a concentration lower than MIC showed a marked elongation of the cells, which indicated that the effects of sulbenicillin was enhanced by dibekacin. These findings suggested strongly that sulbenicillin and dibekacin act on cell wall constituents and that their effects were complementary and synergistic.     

Electron microscopy of early cytoplasmic changes due to influenza virus

Recently improved methods for visualization of thin tissue sections by electron microscopy have been applied to the study of early changes in the bronchial epithelium of mice infected by inhalation of aerosols of influenza virus. In confirmation of previous findings by the authors, inclusion bodies have been demonstrated in ciliated and non-ciliated cells of infected bronchial epithelium. In addition to 3 strains of mouse-adapted Type A virus, 2 unadapted strains gave qualitatively the same results. The inclusion bodies were found to be composed largely of particles of a size estimated to correspond to the known size of influenza virus. The viral lesion of the cytoplasm was also associated with linear formations which were thought to be abnormal forms of endoplasmic reticulum. Well developed microvilli were found on the ciliated borders of ciliated cells, but no evidence was found of viral growth in this region.     

Optical scatter imaging detects mitochondrial swelling in living tissue slices

Mitochondrial swelling is observed in neuronal injury and is a key event in many pathways to cell death. Currently, there is no technique for directly measuring mitochondrial size changes within living tissue slices with a field of view of several millimeters. In this paper, we test our hypothesis that Mie light-scatter theory can be used to study mitochondrial swelling in living tissue sections. Using a unique dual-angle scatter ratio (DASR) optical imaging system previously demonstrated to be sensitive to latex particle size changes and N-methyl-D-aspartate (NMDA) treatment of hippocampal slices, we studied mitochondrial swelling induced by 500 microM NMDA treatment of hippocampal slices. We observed a strong (R(2) = 0.73) and significant (P < 0.000005) correlation between the electron microscopy-determined diameters of swollen, intact mitochondria and the DASR imaging. We examined the robustness of the technique by evaluating the correlation between the dual-angle scatter ratio and the diameter of the dendrites, observed to swell, in NMDA-treated slices and found no correlation (R(2) = 0.06). The advantage of DASR imaging over electron microscopy or other methods of studying mitochondrial swelling is the sensitivity of DASR imaging to mitochondrial swelling over a large field of view (>9 mm(2)) in an intact tissue slice. This novel technique may allow for the study of regional changes in mitochondrial swelling and recovery as sequential events within a single specimen. This technique will eventually be useful in studying the efficacy of stroke and other disease therapies targeting mitochondrial swelling.     

The use of acoustic microscopy for biological tissue characterization

A system of transmission raster acoustic microscope with an ultrasound frequency of 450 MHz has been designed to investigate biological tissues and comparative analysis of their optical and acoustic images. The possibility of obtaining the contrast acoustic images of nonfixed, nonstained biological tissues and viscoelasticity measurements in microscale was demonstrated.