Expansion microscopy for mapping densely packed platelet receptors

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. Fluorescence imaging of platelet receptors, however, is tricky as high density and abundance makes platelet receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Thus, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We have demonstrated that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. For example, we have revealed the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting. Currently, we will use ExM together with different imaging techniques including dSTORM and Structural Illumination Microsocopy to map the localization and distribution of other platelet receptors within the platelet plasma membrane, as well as its association with other receptors and cytoskeletal components to identify the complete picture of the receptor triggered adhesiveness of platelets.