Super-resolution imaging of cell death in Drosophila tissues via expansion and pan-expansion microscopy

Cell death is essential for the survival and homeostasis of nearly all organisms. Programmed cell death (PCD) originally described the reproducible elimination of specific cells during metazoan development. Apoptosis, the best-studied form of PCD, is characterized by distinct morphological, cellular, and biochemical hallmarks. However, the identification of alternative cell death pathways has highlighted the need to clearly distinguish among them, especially when exploring new paradigms of developmental alternative cell death pathways. Furthermore, the discovery of numerous non-lethal functions of apoptotic caspases, collectively termed non-lethal caspase-dependent processes (CDPs), has underscored the importance of differentiating apoptosis from CDPs within tissues in vivo. Morphological analysis remains essential for differentiating death pathways and for determining whether cells are dying or surviving, yet such analyses traditionally relied on electron microscopy, which is labor-intensive, costly, and technically demanding. Expansion microscopy (ExM), a technique that physically enlarges biological samples isotropically, has transformed light microscopy by enabling super- and ultra-resolution imaging using standard fluorescence or confocal platforms. Here, we describe two ExM-based protocols optimized for, but not limited to, Drosophila tissues: ExM, achieving ∼4× expansion, and pan-ExM, yielding ∼12-16× expansion. ExM supports super-resolution imaging of immunofluorescent labels, whereas pan-ExM provides access to ultrastructural features, albeit with limited capacity for specific protein detection. We also compare apoptotic and non-apoptotic cell death morphologies before and after ExM and pan-ExM. Given the growing diversity of ExM strategies, this chapter offers a comprehensive introduction to these rapidly advancing methodologies.