Imaging Endocytosis Dynamics in Health and Disease.

Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, guarantees plasma membrane homeostasis, and generates intracellular signaling cascades. Moreover, it plays an important role in dead cell clearance and defense against external microbes. Finally, endocytosis is an important cellular route for the delivery of nanomedicines for therapeutic treatments. Thus, it is not surprising that both environmental and genetic perturbation of endocytosis have been associated with several human conditions such as cancer, neurological disorders, and virus infections, among others. Over the last decades, a lot of research has been focused on developing advanced imaging methods to monitor endocytosis events with high resolution in living cells and tissues. These include fluorescence imaging, electron microscopy, and correlative and super-resolution microscopy. In this review, we outline the major endocytic pathways and briefly discuss how defects in the molecular machinery of these pathways lead to disease. We then discuss the current imaging methodologies used to study endocytosis in different contexts, highlighting strengths and weaknesses.

Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue.

BACKGROUND: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. METHODS: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. RESULTS: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. CONCLUSIONS: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.