From background to foreground: secondary antibodies coupled to lipophilic ATTO dyes enable high-density membrane labeling in super-resolution and expansion microscopy

Classical immunolabeling approaches can achieve homogeneous and continuous labeling of cellular membranes and organelles at wide-field and confocal resolution. In super-resolution and expansion microscopy, however, the lack of high-density labels hampers the localization of membrane proteins and protein complexes within their membrane context. Here we show that secondary antibodies coupled to the lipophilic dyes ATTO 647N or ATTO 550 brightly label the nuclear envelope, mitochondria, and endoplasmic reticulum of fixed, permeabilized cells, and that graded labelling intensities allow selective visualization of organelles and precise segmentation of mitochondria. Using state-of-the-art super-resolution and expansion microscopy, we achieve high-density labelling of nuclear and mitochondrial membranes, with targeting and density comparable to existing membrane-labelling approaches and a signal that can be further amplified with additional secondary antibodies. Finally, we show that these dye-conjugated IgG allow to resolve mitochondria-ER contacts and mitochondrial ultrastructure as well as precise visualization of the nuclear envelope and its invaginations. This study demonstrates that secondary antibodies conjugated to lipophilic fluorophores represent stable, convenient and affordable tools for organelle visualization in conventional microscopy and for high-density labeling of membranes in super-resolution and expansion microscopy.