There is a long-standing interest in studying viral fusion from a biophysical perspective to dissect the underlying interplay between proteins and lipids involved in the process . In a bottom-up biophysical model, one can precisely control the composition of the system to ascertain which components are essential to a process and quantify their molecular contributions. Fusion of virus particles with lipid vesicles, sometimes referred to as pseudo-infection, is a commonly described model of viral cellular entry under controlled conditions (pH, temperature, time, target membrane composition and curvature) . Since viral membrane fusion is an inherently heterogeneous process, observation of a large number of such events on a single particle level can reveal pathways and mechanistic details that may remain obscure in ensemble studies . Until now, diffraction-limited fluorescence microscopy and cryogenic transmission electron microscopy (cryo-TEM) have been the preferred methods for studying viral fusion on a singleparticle level . While fluorescence microscopy provides information on fusion kinetics, cryo-EM provides a direct visualization of the fusion intermediates over a population. A. Schematic representation of the steps leading to the cellular entry of influenza virus. Fusion of a virus particle with endosomal membrane is triggered by acidification of endosomes (marked in a dashed rectangular box). B. Schematic representation of fusion of influenza virus particles with target vesicles tethered to a passivated and functionalized glass surface (pseudo-infection). The target vesicles encapsulate a fluorogenic nucleic acid-binding dye. In the first step, the virus particles bind to the target vesicles via sialic acidcontaining lipid (GD1a) receptors (two molecules shown for simplicity). Fusion between virus particles and vesicles is triggered by a drop in pH. Upon opening of the fusion pore, the nucleic acid-binding dye molecules come in contact with the viral ribonucleoprotein (vRNP) complexes and turn on in fluorescence. This step is marked with a dashed rectangular box to highlight the analogy with endosomal membrane fusion depicted in part A.