SUMMARY Background Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences. Objectives Our aim was to address how the structural organization of α-granules supports their dynamics. Methods To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image almost entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM). Results and Conclusions In resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations, i.e., macro-homogeneity. In resting platelets the incidence of tubular granule extensions was low, ~4%, but this increased by more than 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by ~3-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for timed release of α-granule proteins.

Obesity affects more than 650 million adults worldwide and is a major risk factor for a variety of serious comorbidities. The prevalence of obesity has tripled in the past forty years and continues to rise. Eosinophils have recently been implicated in providing a protective role against obesity. Decreasing eosinophils exacerbates weight gain and contributes to glucose intolerance in high fat diet‐induced obese animals, while increasing eosinophils prevents high‐fat diet‐induced adipose tissue and body weight gain. Human studies, however, do not support a protective role for eosinophils in obesity. More recent animal studies have also reported conflicting results. Considering these contradictory findings, the relationship between eosinophils and obesity may not be unidirectional. In this mini‐review, we summarize a recent debate regarding the role of adipose tissue eosinophils in metabolic disorders, and discuss local and systemic effects of eosinophils in obesity. Given that adipose eosinophils play a role in tissue homeostasis, more research is needed to understand the primary function of adipose tissue eosinophils in their microenvironment. Therapeutic interventions that target eosinophils in adipose tissue may have the potential to reduce inflammation and body fat, while improving metabolic dysfunction in obese patients.

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

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