Corrected super-resolution microscopy enables nanoscale imaging of auto-fluorescent lung macrophages

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signalling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly auto-fluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove auto-fluorescent noise from Stochastic Optical Reconstruction Microscopy (STORM) datasets. Simulations identified a moving median filter as an accurate and robust correction technique. Using this, we were able to visualise lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. We further show that the tetraspanin and extracellular vesicle (EV) marker CD81 appears in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages, likely marking the secretion of extracellular vesicles. Moreover, this correction method for super-resolution microscopy is widely applicable to other challenging biological samples.