ID-1-P-5720 FIB/SEM and correlative ArraySEM - NanoSIMS study on toxic internalization of hemoglobin by endothelial cells

Free hemoglobin (Hb) forms under oxidizing conditions large protein aggregates, which are heavily and actively taken up by endothelial cells: Membrane ruffles reach out to grab these particles. Several membrane lamella try to wrap them independently and pull them into the cytoplasm. In the final toxic stage the aggregates fill up the cell volume, just separated by thin lamellas. As several internalization processes take place simultaneously, the definition of extracellular versus intracellular space is sometimes difficult and only 3D analysis by targeted FIB/SEM can help to answer the “in-out” question. While lamella formation at the cell surface is clearly visible on SEM-images of freeze dried cells, the internal membrane channels & cavities show up only in the FIB/SEM-cross section. The tomographic data allows to virtual blend out the hemoglobin particles for studying membrane features.

The hemoglobin precipitates are showing up in the micrographs as heavily electron dense particles. To finally proof their nature and off-spring from hemoglobin, elemental mapping for Fe was performed. While EDX, EELS and ToF-SIMS failed to detect iron in these protein-complexes due to sensitivity limitations, imaging mass spectrometry (MS) maps and local measurements became possible with a dedicated NanoSIMS tool below a Fe-concentration of 0.5mM +/- 0,03mM.

Endothelial cells in a body form normally the walls of blood vessels; in culture they are flat adherent cells, growing tight and overlapping. In order to study Hb toxicity, primary human endothelial cells were grown up to confluency on cover slips. The cultures were incubated under oxidizing conditions (GOX) with glucose and 2mg/ml hemoglobin. Under these conditions Hb aggregates heavily. The aggregates get internalized by the endothelial cells. Aggregate formation and uptake was stopped with 2,5% glutaraldehyde/PBS, followed by osmification, dehydration, resin impregnation & polymerization (thin layer plastification) and montage. By keeping the resin layer as thin as possible, hemoglobin precipitates on cells could easily be spotted in the SEM by their prominent topography and FIB/SEM tomography was performed at these preselected spots for a detailed 3D-view on internalization. Image processing allowed to uncover the membrane structures at the internalization site in silico.

Microtome ultrathin sections of block embedded cell were deposited onto ZnO-coated glass slides for SEM observation to define an area of interest for final mass spectrometry analysis. With a CAMECA NanoSIMS we were able to detect Fe (iron) within these hemoglobin aggregates and other cellular ions i.e. phosphate or chloride at subcellular resolution and physiological concentration.