Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

Abstract

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O ). Nox-generated O is rapidly converted to other ROS. Free radical-forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H O generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3'-diaminobenzidine-tetrahydrochloride (DAB) and 0.01% H O . A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).

ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life

“…1 c) that visualize cancer drugs with EELS (Mhawi 2009 ) or stains that are deposited in a specific way depending on probes like the DAB-lanthanides discussed before (Adams et al 2016 ). Similar success with cerium-DAB that is specifically phagocytosed and subcellularly analyzed with EDX and EELS has recently been demonstrated (Moriguchi 2018 ).…”