Degranulation Processes via Electron Microscopy
 
Changes in mast cells with regard to their structure and morphology during degranulation have been studied with several histamine-releasing agents (Bloom 1967, Lagunoff 1973). Compared with untreated cells where all of the granules appear homogenously electron-dense, treated cells were composed of both unaltered and altered granules where the latter appeared to be less dense and separated from their perigranular membrane. The inner structures of these granules appeared as filaments composed of fine particles, leading to the reduction in electron density (Lagunoff 1973).  These less-dense granules appeared to localise near the periphery of the cell (although many inner granules were also involved in the degranulation process) and enclosed in larger vacuoles of cytoplasmic matrix that open up to the outside of the cell (Bloom 1967). However, this extracellular opening was not detected in mast cells treated with polymyxin B sulfate (Lagunoff, 1973). Altered granules were detected extracellularly, indicative of the release of granular contents (histamine) upon stimulation.  An electron-lucent space in between the granules and the perigranular membrane was also evident, mirroring the observation by Smith (1957). At high magnification, adjacent perigranular membranes came into contact with each other forming a five-layer membrane (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 might be involved in histamine release that differs from the exocytosis model  (Lagunoff, 1973).      
The degranulation response varies across mast cell types and the histamine-releasing agents. Certain chemical mediators – polymyxin B sulfate, compound 48/80, bee venom – produce similar morphological changes \cite{4179625} \citep{Bloom_1967}\citep{4158508} in the skin, subcutis, and peritoneal mast cells. Other chemicals such as n-decylamine resulted in a more extensive change in mast cells  (Bloom 1967). Both plasma and perigranular membranes disintegrated and the less dense granules were randomly distributed among electron-dense granules throughout the cytoplasm. At the opposite end of this spectrum, gut mucosal mast cells, which exhibited the aforementioned morphological differences as compared to untreated peritoneal mast cells, were unaffected when treated with compound 48/80, suggesting functional differences exist between mast cells isolated from different sites.      
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 \citep{Chi_1976}. In unstimulated cells, the granules appeared as bulges with evenly distributed particles within the boundary of the bulges (intramembrane particles). In cells incubated with polymyxin B, there were patches in the bulges where the intramembranous particles were absent. These particles appeared to be concentrated at the base of the bulges instead. A pore was also observed that opened into a large channel corresponding to the fusion of vacuoles in mast cells that were fixed at a later time-point after stimulation. Intramembranous particles were detected near the lining of the membrane of the channel, indicative of granular release \citep{Chi_1976}
Corroborative findings regarding unstimulated and stimulated mast cells facilitated investigative studies on factors that may induce mast cell degranulation. A clinical study by \citet{Casale_1984}  investigated the applicability of opiates to be used as a routine test for dermal hypersensitivity in the clinic. Electron micrographs of mast cells obtained from healthy subjects that were injected intradermally with endogenous opioids and opiates were assessed in addition to the wheal-and-flare skin test. Positive results from this test – the formation of wheals and flares – correlated with features of degranulated mast cells as established in literature; this indicated that mast cells could be stimulated by opiates and endogenous opioids through the engagement of opiate receptors. Another study showed the role of skin mast cells in neuroinflammatory skin diseases such as psoriasis and atopic dermatitis where the conditions are induced or worsened by stress \cite{10469524}. A mouse model of acute immobilisation was used to trigger the release of corticotropin releasing hormone (CRH) and neurotensin. Mast cells from the stressed animal exhibited the characteristics of degranulation, which were not apparent when the animals were pre-treated with anti-CRH and neurotensin receptor antagonist. Results from the micrographs augmented the other experimental read-outs, indicating that mast cell degranulation may be implicated in human neurogenic skin disorders (Singh 1999). 
Segregation of Lyn from the FcεRI β clusters was observed in activated cells while Syk appeared to colocalise with the FcεRI β
on These anti-DNP IgE-primed cells exhibited clusters of FcεRI β composed of more than 10 particles when activated with DNP-BSA, which was larger than the 2-3 particle clusters that were mostly observed in resting cells. The distribution of Lyn was similar in resting cells and these clusters often localised with FcεRI β. In contrast, Syk formed individual clusters or appeared as singlets, separated from the FcεRI β clusters. In activated mast cells, the co-localisation of FcεRI β and Syk gold particles was observed, whereas Lyn appeared to aggregate at the periphery of these Syk-FcεRI β clusters, forming a gold string. An insight into the membrane topography and behaviour of the IgE receptor and its associated proteins upon activation was achieved via high resolution TEM.