AFM-STED correlative nanoscopy reveals a dark side in fluorescence microscopy imaging

It is known that the presence of fluorophores can influence the dynamics of molecular processes. Despite this, an affordable technique to control the fluorophore distribution within the sample, as well as the rise of unpredictable anomalous processes induced by the fluorophore itself, is missing. We coupled a stimulated emission depletion (STED) microscope with an atomic force microscope to investigate the formation of amyloid aggregates. In particular, we studied the in vitro aggregation of insulin and two alloforms of β amyloid peptides. We followed standard methods to induce the aggregation and to label the molecules at different dye-to-protein ratios. Only a fraction of the fibrillar aggregates was displayed in STED images, indicating that the labeled molecules did not participate indistinctly to the aggregation process. This finding demonstrates that labeled molecules follow only selected pathways of aggregation, among the multiple that are present in the aggregation reaction.