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As You Like It, Part 3, is a continuation of the lectures given by the author (Ultra Path VIII and Ultra Path IX). It is a critical historical review of topics of interest to electron microscopists, attempting to show what went wrong and perhaps also why. The topics chosen this time demonstrate the prominent role electron microscopy has played in elucidating the diverse ways in which calcification can occur. The classic concept of dystropic and metastatic calcification is now inadequate to explain all observed phenomena. The electron microscope shows that calcification occurs in many different intracellular and extracellular sites and that each has its own morphology and etiology. Thus, a new classification based on ultrastructural morphology is born, but few seem to be aware of it. The author examines the ubiquitous but not too well-known phenomenon of cell remodeling, which often, but not always, leads to calcification. Topics are presented under the following headings: (1) matrical lipidic debris, (2) spherical microparticles, (3) matrix vesicles, (4) intramitochondrial calcification, (5) intralysosomal calcification, (6) calcification of collagen, (7) calcification of elastic fibers, and (8) calcification of secretory products.

As You Like It, Part 2, is a continuation of the lecture given at UltraPath VIII in 1996. It is meant to be a critical historical review of some topics of interest to electron microscopists that will show what went wrong and perhaps also why. Topics are presented under the following headings : (1) Amianthoid Fibers and Giant Collagen Fibrils, (2) Ribosome-Lamella Complex, (3) Globular Filamentous Bodies, and (4) Siderosomes, Hemosiderin vs. Ferritin.

Despite many studies on melanosis thyroidi, there is no consensus concerning the nature of the pigment or its pathogenesis. Here the past literature on this subject is reviewed and results are reported on a case of melanosis thyroidi that provides fresh insight into the nature of the pigment and its genesis. Briefly, the conclusion is that while a few of the pigment granules are classic lipofuscin granules (i.e., single-membrane-bound structures containing electron-dense material in the form of particles, granules and masses, and electron-lucent lipid droplets), a majority of the pigment granules also contain colloid, which is easily identified by its particulate substructure similar to the colloid in the lumen of follicles. Thus, these pigment granules are, in fact, ambilysosomes, where degradation of both endogenous (organelles) and exogenous (colloid) material occurs. Several phagosomes containing colloid were also seen in the thyroid epithelial cells.

I have developed something of a reputation for criticizing freely and frequently at our meetings and in my writings. You say you like this and find it useful and entertaining. As you like it, you are welcome to more of it. My comments and criticisms are presented under the following headings: (1) criticize at your peril; (2) how it all started (unjustly accused!); (3) abbreviations (a source of perennial aggravation, confusion, and waste of time); (4) mysterious bodies in mesotheliomas; (5) call a crystal a "crystal," not a "crystalloid"; (6) electron microscopy-a study of osmium artifacts; (7) length-to-diameter ratio of microvilli (mission impossible); (8) lamellar bodies (a popular but debased term); (9) amyloid filaments, not fibers; (10) filaments and microtubules do not branch; (11) there is no such thing as pseudomelanosis; (12) botched histochemistry (just about every gastrointestinal tract pigment was misdiagnosed by histochemistry); (13) intranuclear Russell bodies, not "Dutcher bodies"; and (14) nuclear pores and virus-like particles (a new development in an old farce).

A presumably new structure that we have called "globular smooth-tubule aggregate" was found in the cytoplasm of chondrocytes of 4 thanatophoric dwarfs. To the best of our knowledge these sharply delineated, rounded, sieve-like bodies of distinctive morphology have not been reported to occur in any other site or situation. Hence these structures may be of some diagnostic value. Continuity between the rough endoplasmic reticulum and smooth tubules in the aggregates was detected. Therefore it is suggested that smooth tubules in these aggregates derive by growth and extension from the rough endoplasmic reticulum.

The lengths of desmosomal profiles were measured in sections of tumor tissue from cases of mesothelioma, adenocarcinoma, squamous cell carcinoma, thymoma, and meningioma. Giant desmosomes (length of profile 1 micron or greater than 1 micron) were found in all the above-mentioned tumors except adenocarcinomas. The largest desmosomal profile in adenocarcinoma was approximately 0.8 micron long; the largest in mesothelioma was approximately 2 microns long. Our observations suggest that one of the ways in which giant desmosomes arise is by growth and fusion of adjacent desmosomes. Giant desmosomes may at times help in distinguishing mesothelioma from adenocarcinoma, but this is a rather rare phenomenon. In this study giant desmosomes were found in only 2 out of 10 cases of mesothelioma.

It is now apparent that light microscopy and histochemistry failed to identify correctly the nature and composition of pigments in various gastrointestinal tract melanoses. In most instances it was thought that the pigment was melanin or a melanin-like substance. Electron microscopy (EM) and electron-probe energy dispersive x-ray analysis have rectified these errors and have shown the following: in melanosis coli the pigment granules contain lipofuscin; in melanoses ilei the pigment granules may contain either silicates and titanium or hemosiderin; and in melanosis duodeni the pigment granules contain iron sulfide. In melanosis esophagi it is not clear what the pigment is; it could be melanin or lipofuscin.

This study was undertaken to examine the morphological basis for noncalcific cusp thickening in 20 standard-profile Ionescu-Shiley bovine pericardial cardiac valvular bioprostheses (16 aortic, four mitral) with primary valve failure due to cusp tears. The patients included 17 males and three females, with a mean age of 52.0 years; their valves had been implanted for a mean of 105.8 months. All cases had variable degrees of "plasma insudation" without correlation to identifiable patient parameters. This was accentuated in individual valves in the cusp with the most extensive tears. In addition, the three valves with the thickest cusps were grossly distorted by massive deposits of amyloid (identified as AL-amyloid in two index cases studied). Two other valves had incidental amyloid deposits, identifiable only by electron microscopy. Amyloid involvement of pericardial bioprostheses has not been previously reported. The literature pertaining to amyloid involvement and plasma insudation of bioprostheses is reviewed.

We report here a case of melanosis ilei where grossly visible greyish black pigmentation of the ileal mucosa was discovered at autopsy. Light microscopy showed Prussian blue positive pigment granules in macrophages in the lamina propria forming the core of villi. Ultrastructural examination showed that the pigment granules had the morphology of siderosomes which are known to be the electron microscopic equivalent of the haemosiderin granules seen with the light microscope. We know of no previous report where pigmentation of the ileal mucosa was ascribed to the presence of haemosiderin.

Electronmicroscopy and electron probe energy dispersive X-ray analysis studies have substantially contributed to our understanding of the various gastrointestinal tract melanoses. The nature of the pigment granules which occur in the various melanoses is discussed; their pattern of distribution in melanosis coli, melanosis ilei, melanosis duodeni and melanosis oesophagi is summarized and current knowledge of the aetiology and pathogenesis of these conditions is reviewed. Brief mention is also made of other examples of lipofuscin pigmentation, and a case of haemosiderosis ilei is described.

Two cases of melanosis ilei were studied, in which grossly visible blackish pigmentation of the ileal mucosa was incidentally discovered at autopsy. Light microscopy showed that the pigment granules lay within macrophages in atrophic Peyer's patches. Ultrastructural studies showed that the pigment granules were heterolysosomes containing crystalline material, particles, granules and, occasionally, lipid droplets. The morphological similarity between these pigment granules and granules in pulmonary macrophages was established through ultrastructural studies of pulmonary lymph nodes obtained during routine autopsies. These data, plus results of past electron-probe X-ray analytic studies by us and others, leads us to conclude that the pigment granules in melanosis ilei contain exogenous material derived from inspired and ingested materials.

Foreign materials are often seen by light microscopy in surgical or autopsy specimens sent from the cardiovascular clinical services. This paper describes the range of materials that may be recovered in such specimens and provides a pictorial guide to aid in their identification. The spectrum of medical and nonmedical foreign materials that may be encountered in the cardiovascular system is discussed., (Copyright © 1993. Published by Elsevier Inc.)

Until now microvillus-matrix associations have been reported to occur only in mesotheliomas and hence this phenomenon is thought to be of value in distinguishing mesothelioma from pulmonary adenocarcinoma. We report here the occurrence of microvillus-matrix associations in two pulmonary adenocarcinomas which reduces the value of this phenomenon in the differential diagnosis of these tumors.

Siderosomes (i.e. single membrane-bound lysosomal bodies containing haemosiderin) were produced in the liver and muscle of rats by injections of iron dextran. Electron-probe X-ray analysis was executed on siderosomes in cryosections of quick-frozen fresh unfixed tissues (liver and muscle) and sections of Epon-embedded tissues. There were no statistically significant differences between the ratios of iron:phosphorus and iron:sulphur in these two types of preparations. Hence it is concluded that there is no significant loss or gain of phosphorus or sulphur during preparation of tissues for Epon embedding. The results confirm past belief that so little phosphorus or sulphur is present in siderosomes that haemosiderin is best regarded as ferric hydroxide oxide. A new finding in the present study was the demonstration of small amounts of potassium in siderosomes in cryosections. It seems that potassium is lost (leaches out) from siderosomes during preparation of tissues for Epon-embedding.

Chlorotriethylphosphine gold (SK & F 36914) administered orally to rabbits produced aurosomes in the synovial membrane. These aurosomes were similar to aurosomes produced by parenteral or intra-articular injections of soluble gold salts such as sodium aurothiomalate and aurothioglucose. This study shows, (1) that an orally administered gold compound is capable of producing gold deposits in the synovial membrane, and (2) that such compounds may have a future in the treatment of rheumatoid arthritis.

The synovial membranes of 5 young rabbits about 6 weeks old and 5 older rabbits about 3-4 years old were studied with the light and electron microscopes, with a view to discovering any age-associated changes that might occur. Several age-associated changes were noted. These included: (1) a reduction in the overall population of synovial intimal cells; (2) a statistically significant reduction in type B cells and a relative increase in type A cells; (3) the emergence of atrophic cells poorly endowed with organelles; (4) decreased vascularity; and (5) fibrosis.

Intra-articular administration of colloidal gold produced aurosomes which contained spherical electron-dense granules and also often some very fine electron-dense particles. Intra-articular administration of aurothioglucose produced aurosomes of a morphology similar to that reported to occur after administration of sodium aurothiomalate. These aurosomes produced by soluble gold salts are characterised by particle and granule-studded membranous formations which present in profile as rod-like and curled filamentous structures. On the basis of morphological differences two types of aurosomes should be recognised, the "Type I or granular aurosome" and the "Type II or membranous aurosome".

A serial section study was executed on a lymph node from a case of AIDS (acquired immune deficiency syndrome) to determine the true three-dimensional morphology and mode of genesis of tubular confronting cisternae which are found in the lymphocytes in this condition. Serial sections from over a hundred such structures show that these are tubular structures and that the 'test-tube and ring-shaped' forms described in the literature are no more than profiles one expects to see when a tubular structure is sectioned. The view at times expressed in the literature that these structures are truly test-tube-shaped (i.e. one end closed) appears to be erroneous, for our serial section study did not reveal a single instance where one end was closed; all were tubular structures with both ends open. We found that tubular confronting cisternae develop by a process of twisting and spiralling of ribbon-shaped confronting cisternae, and that at one stage of development there is a spiralling groove on the wall of this structure. Microtubuloreticular structures were seen in several lymphoid cells, sometimes they were found in cells containing tubular confronting cisternae. At times microtubules (similar to those seen in microtubuloreticular structures) were seen lying within and/or outside the profiles of dense lamina of tubular confronting cisternae.

A light and transmission electron microscopical study of 6 to 8 months old rabbit semilunar cartilages has shown that the cells in this tissue resemble chondrocytes more than fibroblasts. The prominent organelles in these cells were rough endoplasmic reticulum and the Golgi complex. An unusual finding was the occurrence of filamentous material in Golgi sacs and vesicles, and collagen fibrils within smooth membrane-bound tubular structures, apparently within the cells. Collagen fibrils forming fibres, fibre bundles and lamellae constituted the major component of the menisci. Protein-polysaccharide particles and associated fine filaments were found in the interfibrillary matrix and in the sparse territorial matrix adjacent to the chondrocytes. Numerous immature elastic fibrils, and rare mature elastic fibres with an electorn-lucent amorphous core, were also found in the general matrix amongst the collagen fibrils.

A case of oncocytic carcinoid of the lung was studied with the electron microscope. This rare tumour is composed of cells acceptable as neuroendocrine cells, oncocytes and cells of an intermediate morphology. Our studies lead us to believe that this tumour develops by oncocytic transformation of neuroendocrine cells in a carcinoid. Two new findings reported in this study are: 1) the occurrence of filamentous inclusions in mitochondria; and 2) calcified spherules which appear to develop in material secreted by tumour cells.

Aurosomes are lysosomal bodies containing gold. Colloidal gold produces aurosomes containing spherical electron-dense granules. Soluble gold salts produce aurosomes containing lamellar, filamentous and rod-like profiles studded with particles and granules. This morphological pattern is quite distinctive, and is not affected or altered by which particular soluble gold salt is administered, by which route it is administered or in which species or cell type the aurosome occurs. In the skin of patients treated with soluble gold salts the characteristics electron-dense formations indicating the presence of gold are often found in compound melanosomes and other lysosomes in the dermal macrophages; while in the synovial membrane these characteristic electron-dense deposits are seen in the numerous lysosomes that develop in the rheumatoid synovial membrane. Thus it would appear that while aurosomes may vary somewhat in morphology, the electron dense contents indicating the presence of gold have a fairly constant morphology.

Intra-articularly administered sodium aurothiomalate (Myochrysine) produced aurosomes containing characteristic electron dense contents (indicating the presence of gold), in the chondrocytes of rabbit articular cartilage. At first the aurosomes were bounded by a membrane but later the electron dense contents were seen lying free in the cytoplasmic matrix. Such deposits were detectable up to 14 months after injection of Myochrysine but none were found at later time intervals (18 months and 2 years). There was a reduction in the population of superficial chondrocytes (Zone I) while those in deeper zones (Zones II and III) showed an increased content of intracytoplasmic filaments. It is thought that these are regressive or degenerative changes produced by gold.

In a case of chronic lymphocytic leukaemia we found that a few (less than 5%) of the circulating leukaemic lymphocytes contained inclusions of a type which do not appear to have been reported in the literature. The inclusions presented as solitary tubules or aggregates of parallel tubules lying within dilated cisternae of the rough endoplasmic reticulum and the perinuclear cistern. Some of the tubules were bounded by a single wall, others by a double wall. We also found a tubule bounded by three walls and another where a segment of the tubular profile was triple-walled and the remainder double-walled. We are inclined to think that the walls of these tubules are composed of membranes. The aetiogenic agent or agents responsible for the production of intracisternal tubules and their mode of formation are obscure.

Scanning electron microscopic studies revealed numerous humps on the surface or articular cartilage from a 7 years old boy and from 1 month old rabbits. This shows that the surface topography of articular cartilage in young individuals is different from that of older individuals (human and rabbit) where the surface is beset by numerous pits. In 2 months old rabbits an intermediate situation was witnessed, for both shallow pits and occasional humps were present. Ridges and undulations were not seen on the articular surface of the 7 years old boy, except near the cut edge of the specimen. They were, however, found on air-dried specimens of 1 month old rabbit cartilage, but were absent from specimens dried by the critical-point method. The collective evidence supports the idea that ridges and undulations are not a constant feature of the normal articular surface, but that such features are artefactual, a typical or pathological.

An increase in the number of hepatocellular lysosomes is detectable with the electron microscope in rats bearing a carcinogen-induced sarcoma but not in rats bearing a transplanted Walker tumour. Biochemical studies show an increase in lysosomal enzymes in the liver of both groups of rats, but the increase in the rats bearing a carcinogen-induced sarcoma was greater than that in rats bearing a Walker tumour. This difference is explained by the fact that only gross increases in lysosomal population are likely to be detected in the small samples examined with the electron microscope, but by biochemical methods whereby many grams of tissue are analysed, finer changes may be detected. The collective data to date indicate that an increase in hepatocellular lysosomes and/or lysosomal enzymes occurs in rats and man bearing a variety of tumours and to this there is, as yet, no known exception.

A myofibroblastoma occurring in the abdominal cavity of a 15 year old boy is described. This tumour was diagnosed as a low grade sarcoma by light microscopy but electron microscopy showed that the tumour was composed almost entirely of myofibroblasts and a few macrophages. Intermediate forms between myofibroblasts and macrophages were not seen nor were any fibroblasts seen in the main tumour mass. Total excision was impossible because the tumour had trapped loops of bowel and was adherent to the abdominal organs. The patient died of cachexia and haemorrhage but there were no distant metastases nor was there any marked infiltration of the abdominal organs. This case and a review of the literature shows that myofibroblastomas are locally aggressive tumours which do not metastasize and that if total excision is possible an uneventful recovery can be expected.

A morphometric study was carried out to assess the thickness of the fibrous lamina in the nuclei of cells adjacent to tears in semilunar cartilages and in cells from relatively normal uninjured parts of semilunar cartilages. Comparisons of mean thickness of the lamina showed a statistically significant thickening of the lamina at the site of injury. This observation supports the growing body of evidence indicating that the fibrous lamina is not a static structure of fixed dimensions for a given cell type but a dynamic structure whose thickness varies as a result of altered physiological or pathological states.

The articular surface of the femoral condyle of kittens (2 days to 2 months old) and cats (6 months to 20 months old) was studied by examining air dried and critical point dried specimens in the scanning electron microscope. The surface of kitten cartilage was found to be populated by innumerable humps which were more prominent in air dried than in critical point dried specimens. Undulations and ridges were seen on air dried kitten cartilage, which was markedly shrunken and distorted, but undulations and ridges were absent from critical point dried specimens where shrinkage was more modest or even undetectable. The surface of the articular cartilage of 12 months and 20 months old cats was populated by innumerable pits. A rare hump in a pit was seen in specimens from the 12 months old cat, but not from the 20 months old animal. In 6--8 months old cats an intermediate situation prevails, for in some specimens pits were present on the posterior part of the condyle but humps and humps in pits were present on the anterior aspect. This study shows that the surface of young articular cartilage is populated by humps, but as the cartilage matures these formations are either transformed into pits or replaced by pits.

Haematomas were produced in rabbits by subcutaneous injection of autologous blood. Clotting and marked lysis of erythrocytes was noted in these haematomas, but there was no evidence of fragmentation of erythrocytes prior to or after ingestion by macrophages as has been reported in other sites such as the peritoneal cavity and the joint cavity. The phagocytosis of intact erythrocytes, lysed erythrocytes and haemoglobin led to the formation of three main types of lysosomal bodies; (1) myelinosomes containing whorled osmiophilic membranes, (2) siderosomes containing haemosiderin, and (3) myelinosiderosomes containing a mixture of osmiophilic membranes and haemosiderin.

In a largely haemorrhagic and necrotic specimen of Ewing's sarcoma we found banded structures (periodicity of banding about 250 nm) of a type hitherto not known to occur in any normal or pathological tissue. Morphological appearances suggested that these structures had evolved from the fibrin which was always closely associated with them. However, this idea was abandoned because the periodicity of banding of these structures is about ten times greater than the known periodicity of banding of fibrin. These structures have the same periodicity of banding as fibrous long-spacing collagen produced in vitro and about twice the periodicity of banding of fibrous long-spacing collagen found in vivo. However, they lack the fine striations or minor bands of the in vitro fibrous long-spacing collagen. We therefore conclude that the banded structures in this tumour represent a new form of fibrous long-spacing collagen which shares the features of in vitro and in vivo types of fibrous long-spacing collagen.

Giant melanosomes measuring up to about 5.1 micrometers in diameter were seen in the eyes of rabbits with metallic implants. Somewhat smaller giant melanosomes up to about 2.7 micrometers in diameter were seen in the normal eye of a rabbit. Such melanosomes were not found in eyes of rabbits less than a day old. Some giant Stage IV melanosomes appear to derive from giant Stage III melanosomes with an internal structure similar to that in the normal smaller melanosomes, while others appear to be derived by fusion of smaller melanosomes. There may be yet another mechanism of production of giant melanosomes involving 'vesicular' structures but about this one cannot be certain.

Cells in culture were exposed to cis-dichlorodiammineplatinum (II) and platinum-uracil blue. No platinum could be demonstrated in an intracellular location morphologically or by electron-probe x-ray analysis in the case of the former compound. However, platinum was readily demonstrated in phagosomes or phagolysosomes after exposure of cells to platinum-uracil blue.