Studying Mast Cell Morphology by Electron MicroscopyThe limitation of light microscopy in the study of mast cells prompted the employment of other imaging platforms. A platform that was widely used in the early sixties through mid-eighties was the electron microscopy. Using the RCA EMU-2A electron microscope, \citet{Smith_1957} studied mast cells isolated – via centrifugation – from the peritoneal fluid of adult male rodents. The cells were dehydrated with various alcohol concentrations before they were embedded in plastic and gelatin. Because of the poor quality in preserving the structural integrity of the mast cells, the authors attempted to optimise the preparatory procedures by using several fixative agents at different times of fixation as well as varying the pH of the fixative and adding modifications to the polymerisation of the embedding plastic. None of the modifications yielded significant improvements to the quality of the electron micrographs. However, the authors stressed that the mast cells that they obtained appeared similar to those isolated from skin, spleen, and liver that were prepared according to the method developed by \citet{14927794}. The embedded cells were cut into thin sections prior to imaging.In addition to imaging normal mast cells, the effects of total body irradiation and intraperitoneal (IP) injection of heparin-binding agent – toluidine blue and protamine sulfate – and histamine-releasing agent, stilbamidine were investigated. The morphology of normal mast cells was described as observed under the electron microscope: large nucleus occupied by thread-like structures with empty spaces towards the interior, multiple round or oval-like electron-dense bodies with a diameter of 0.5-1.0 µ. Thread-like materials that resemble those in the nucleus were present inside these electron-dense structures (cytoplasmic granules), albeit they were thinner with fine granule-like particles attached to them. In cells treated with the heparin-binding agents and stilbamadine, the size of the granules and the inner structures remained unchanged. Each granule seemed to be surrounded by a halo of clear cytoplasm enclosed and separated from each other by the perigranular membrane. The authors attributed this change in the electron micrographs due to cell swelling as a result of the fluid-filled IP injection. The authors noted that although the treatment caused the cells to be turgid, it did not affect the integrity of the granules. Unlike the untreated and IP-injected mast cells, cells isolated from irradiated rodents had more electron-dense bodies that were much larger than the typical size range of a secretory granule with fewer reticular structures on the inside. The nucleus appeared elongated and deformed and together with the absence of intragranular structures, mirrored that observed under the light microscope, which is suggestive of cells undergoing apoptosis \citep{Smith_1957}. The plasma membrane of rat peritoneal mast cells was reported to exhibit finger-like projections that protrude into the extracellular space \citep{Lagunoff_1972}. This structural component was also observed in rat mast cells in the tongue \citep{Enerb_ck_1974}. However, this microvillus-like folding of the membrane was absent in mast cells observed in the lamina propria of the gut mucosa. In addition to the smooth cell membrane, the morphology of these cells slightly differs from that observed in peritoneal (Lagunoff, 1972) and mesentery (Smith 2006) mast cells. Mucosal mast cells often exhibited irregularly-shaped nuclei with indentations \citep{Enerb_ck_1974}, and have fewer but larger granules as compared to tongue mast cells, which have numerous and fairly-uniform sized granules that are within the size range reported by Smith (1957). The intragranular structures between these two cells were observed to be relatively distinct such that most of the granules in tongue mast cells exhibited a homogeneous fine particulate appearance whereas those in mucosal mast cells were composed of coarse particles. Besides this, other mesenchymal and granular (mainly eosinophils) cell types as well as phagocytic immune cells, were typically found in contact or in close proximity to mucosal mast cells \citep{Enerb_ck_1974}. Lagunoff (1972) noted that these cells, which appeared smaller than peritoneal mast cells, were often found localised near nerves and blood vessels.Following electron microscopic studies on animal mast cells, \citet{Hibbs_1960} attempted to study the morphology of human mast cells under normal/untreated conditions. Tissue biopsies were obtained from digital and abdominal skin and subcutis, as well as the gastric mucosa. The samples were divided for imaging by light and electron microscopy where the tissue specimens were processed as described by Smith (1957) for imaging with the electron microscope. Because eosinophils and neutrophils were easily distinguished, granular cells that were not identified as either eosinophils or neutrophils were assumed to be mast cells. Two morphologically distinct cells were observed: One was spindle-shaped with an elongated nucleus and numerous tightly-packed granules wherein the internal structures were not visually apparent; the other type was round or ovoid with many cytoplasmic granules that were more widely distributed. Unlike the light microscope, at the highest magnification of X50 000 of the objective lens of the electron microscope, the authors were able to observe the intragranular structures in the ovoid cells. They appeared to consist of two components – fine particles and lamellar/scroll-like structures. Variations exist between cells with regard to the composition and localisation of these components such that the granules may be composed of mostly fine particles or lamellar or a mix of both. Similar to the skin and dermis, both cell types were observed in the gastric mucosa specimen. However, only the ovoid cells were found in the specimen obtained from a patient who received intensive adrenocorticosteroid treatment. Furthermore, only the lamellar structure of the granules could be observed; the other component was rarely found in these cells, which lead the authors to the hypothesis that the steroid treatment caused the fine particles to be released from the granules. The study also highlighted another advantage of electron microscopy – it enabled the identification of two cell types as distinct cells, which was not feasible via the light microscope \citep{Hibbs_1960}.Electron microscopic studies of mast cells have been done with ultra-thin sections of tissue specimens and liquid samples from various tissues and mammals. \citet{Smith_2006} compiled a list of studies highlighting the source of mast cells that were utilised to-date and noted that the cells had undergone similar processes prior to imaging, that is the cells were fixed with osmium tetroxide and methacrylate, followed by embedding in plastic/Epon resin, and stained with uranyl acetate. Micrographs from these individual studies of normal mast cells appeared similar across species (Smith 2006), corroborating findings from early pioneering studies (Smith, 1957; Hibbs et al.,1960). For instance, the separation between granules by a two-layer electron-dense structure was seen in mast cells that had undergone imbibition of fluid, corresponding to observations by Smith (1957). In another study, many of the granules were only partially surrounded by the perigranular membrane \citet{Lagunoff_1972} . With reference to a study of isolated mast cells (cite) where the histamine content remained despite the lost of the integrity of the perigranular membrane, Lagunoff (1972) suggested that this membrane may not be part of the granular structure itself nor is it integral to the preservation of granular contents inside mast cells under this experimental condition. However, the intragranular structures showed variability across different mast cell types, and this variation was seen in mesenteric mast cells isolated from untreated rats. Nonetheless, functional differences between mast cells could not be used to explain this variation due to the cells being subjected to the extensive processing prior to imaging, which may have altered the granular state of the cells. Degranulation Processes via Electron MicroscopyChanges in mast cells with regard to their structure and morphology during degranulation have been studied with several histamine-releasing agents. Rat peritoneal mast cells incubated with 0.3 µg/ml of compound 48/80 for 15 seconds at 22ºC showed marked alterations in the cytoplasmic granules (Bloom et al., 1967). Compared with untreated cells where all of the granules appear homogenously electron-dense, these cells were composed of both unaltered and altered granules where the latter appeared to be less dense and separated from their perigranular membrane. The authors also noted the presence of an electron-lucent space in between the granules and the perigranular membrane that was first observed by Smith (1957). The electron micrograph of mast cells incubated with 5 µg/ml of bee venom for 20 seconds at the same temperature showed similar morphological changes – the degranulated mast cells exhibited many less-dense granules near the periphery of the cell, which appeared to be enclosed in larger vacuoles of cytoplasmic matrix that open up to the outside of the cell \cite{Bloom_1967}. These altered granules were also observed extracellularly, indicative of the release of granular contents (histamine) upon stimulation. Mast cells incubated with 0.5 µg/ml of polymyxin B sulfate for 5 minutes at 30 ºC also showed these characteristic changes, albeit there was no extracellular opening of the affected granule-containing vacuoles (Lagunoff, 1973). After exposure to the histamine-releasing agent, the inner structures of affected granules appeared as filaments composed of fine particles, leading to the reduction in electron density. Micrographs in the study showed that not only peripheral granules, but also many inner granules were involved in the degranulation process. At high magnification, adjacent perigranular membranes came into contact with each other forming a five-layer membrane \citep{Lagunoff_1973} and the resulting close proximity in the intergranular space was suggested to result in the formation of the extensive cytoplasmic channel along the cell periphery (Lagunoff 1972). Perigranular membrane at the periphery of mast cells also fused with the plasma membrane; in several micrographs, the fused membrane was connected to an extracellular protrusion that resembled an early pore formation, which the author suggested, might be involved in histamine release that differs from the exocytosis model \citep{Lagunoff_1973}. Treatment with 50 µg/ml of n-decylamine, however, resulted in a more extensive change in mast cell morphology \cite{Bloom_1967}. Both plasma and perigranular membranes disintegrated and the less dense granules were not localised near the periphery of the cell but were randomly distributed among electron-dense granules throughout the cytoplasm. The absence of the vacuole-like structures that were observed with the aforementioned histamine-releasing agents was evident in these mast cells. Other cytoplasmic organelles were also affected whereby the mitochondria appeared abnormal and the endoplasmic reticulum was either enlarged or fragmented \cite{Bloom_1967}. In another study where a different methodology was employed to study the effect of compound 48/80 on tongue mast cells, a relatively similar extent of morphological changes to mast cells was observed. Rats were injected intravenously with 0.2 mg/kg of compound 48/80 prior to obtaining the tongue specimen from which the mast cells had lost the integrity of their cell membrane and their nuclei appeared to be in a pyknotic state. Nonetheless, observations characteristic of degranulation were also reported – coarse particles in the granules showing reduced electron density and the presence of vacuoles containing multiple of these granules. In contrast, this compound had no effect on mucosal mast cells in the duodenum, which showed the same morphology as those observed in untreated tongue and duodenum specimen \citep{Enerb_ck_1974}. In addition to transmission/scanning electron microscopy, freeze-fracture electron microscopy offered a different outlook on mast cell degranulation that complimented the micrographs of stimulated mast cells. Many bulges appeared when peritoneal mast cells were incubated with polymyxin B at 15ºC and 22ºC 2-3 seconds before they were fixed with 4% glutaraldehyde. In unstimulated cells, particles within the boundary of the bulges (intramembrane particles) were approximately evenly distributed. In treated cells, this was replaced by patches in which the intramembrane particles were absent and that these particles appeared to be concentrated at the base of the bulges instead. References ? D. E. Smith. ELECTRON MICROSCOPY OF THE TISSUE MAST CELL. The Journal of Cell Biology 3, 9–14 Rockefeller University Press, 1957. GE PALADE. A study of fixation for electron microscopy.. J Exp Med 95, 285-98 (1952). David Lagunoff. Contributions of Electron Microscopy to the Study of Mast Cells. Journal of Investigative Dermatology 58, 296–311 Elsevier BV, 1972 4112450 Lennart Enerb�ck, PerM. Lundin. Ultrastructure of mucosal mast cells in normal and compound 48/80-treated rats. 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