Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

Abstract

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.

High‐resolution 3D imaging of microvascular architecture in human glioma tissues using X‐ray phase‐contrast computed tomography as a potential adjunct to histopathology

“…To date, optical imaging techniques such as confocal laser endomicroscopy (CLE) or ultrastructural methods such as transmission electron microscopy (TEM) have been available for visualizing microstructures in 3D on micrometer or nanometer scales, but they are limited by the low penetration depth, one of the well-known limitations of these methods. 32,33 To address these limitations, PCCT was applied to develop the glioma tissues, which is a label-free, highresolution imaging modality with excellent soft-tissue contrast and superior penetration depth. As expected, the morphology of microvascular proliferation in glioma tissues was clearly observed via PCCT in this study, confirmed by the corresponding histological results.…”

Modulation of Secretory Lysosomes During NK Cell Education Leads to Accumulation of Granzyme B and Enhanced Functional Potential

“…However, another contributing factor may be LYST-mediated modulation of granules during during the effector response allowing smaller proportions of the large granules to be released, as has been demonstrated for degranulation in mast cells. 79 An outstanding question is how lysosomal fission leads to loss of the granular matrix and granzyme B in hyporesponsive NK cells. Granzyme B can be synthesized and secreted directly through the constitutive secretory pathway.…”

PSF correction in soft x-ray tomography

“…From simple yeast to complex eukaryotic cells. SXT grants an unprecedented contrast and spatial resolution, filling the information gap between light and electron microscopy [33,30,10,5,17,4].…”