Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages

Abstract

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize 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. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 5 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

“…Using quantitative analysis, these methods have been further used to define EV size (Mondal et al, 2019;Zong et al, 2018;Nizamudeen et al 2018;Lennon et al 2019;Sharma et al, 2020) and to quantify protein content (Lennon et al, 2019) and number of localizations of miRNA (Oleksiuk et al, 2015) and DNA fragments in EVs (Maire et al, 2021). Additionally, STED and SMLM have been used to image cellular uptake (Chen et al, 2016;Chen et al, 2018;Polanco et al, 2018;Pfeiler et al, 2019;Toda et al, 2020) and release of EVs (Ambrose et al, 2020) or EV clusters (Valcz et al, 2019). Super-resolution microscopy methods comprise tailored approaches for sample preparation, sample imaging, and data analysis.…”

Heterogeneity in extracellular vesicle secretion by single human macrophages revealed by super‐resolution microscopy

“…Following STORM acquisition, all datasets were background corrected using a custom FIJI (Schindelin et al., 2012) script, as previously described (Ambrose et al., 2020). This removed out‐of‐focus fluorescence and auto‐fluorescence from lung macrophages to improve localisation precision and reconstruction accuracy.…”

“…The corrected dataset was then reconstructed using the ImageJ plugin ThunderSTORM (Ovesný et al., 2014), as previously described (Ambrose et al., 2020; Oszmiana et al., 2016). Datasets were filtered with a B‐spline wavelet filter, approximate localisations calculated using local maximum and sub‐pixel localisations determined using an integrated Gaussian point spread function with maximum likelihood fitting applied over a five‐pixel radius.…”

Uncovering the cytotoxic effects of air pollution with multi-modal imaging of in vitro respiratory models

“…Scale bars are 5 μm in main image and 0.5 μm in zoomed images. Adapted from Ambrose et al ([ 155 ], fig. 7B); copyright 2020, Elsevier Group; .…”

“…These methods can accommodate samples with significantly higher fluorophore labelling density; however, they offer a more modest gain in spatial resolution. STORM has been applied to visualize changes in the nanoscale membrane topology of lung macrophages and the spatial distributions of major histocompatibility complex (MHC) class I proteins, [ 155 ] ( figure 4 b ). Image data showed the formation of membrane projections tipped with these proteins following activation of the cells via Fc receptors by addition of human immunoglobin G antibody.…”