Labelling strategies and expansion factors in ExM: key steps towards super-resolution microscopy

Expansion Microscopy (ExM) enables super-resolution imaging by physically enlarging biological specimens, thereby decoupling spatial resolution from optical point spread function engineering. While effective resolution is often described as optical resolution divided by the expansion factor, this view overlooks a fundamental limitation of ExM: once optical constraints are relaxed, accuracy and fidelity of nanoscale imaging become dominated by fluorescent labelling quality and density, as well as reliable expansion factor determination at the cellular level. In this review, we frame labelling efficiency and expansion factor calibration as inseparable, critical elements of quantitative ExM. We provide a structured overview of strategies to enhance labelling efficiency, including anchoring chemistries, pre-versus post-expansion labelling, signal amplification schemes, and protocol optimization, focusing on nuclear lamina studies as a paradigmatic example. We further review approaches to determine expansion factors beyond macroscopic gel measurements, emphasizing intracellular nanometric rulers like nuclear pore complexes and intrinsic calibration strategies based on deformation mapping, such as GelMap. Finally, we discuss how incomplete or inhomogeneous labelling directly impacts metric accuracy under well-characterized expansion conditions, and provide practical guidelines and reporting recommendations for quantitative ExM. Together, this review aims to guide the transition of ExM from qualitative visualization toward robust quantitative molecular nanoscopy.