Step-wise morphological change of influenza vaccine particles. The osmotic shrinkage behavior of the particles (a split vaccine, b WIV vaccine) was comparatively investigated by applying a hyper-osmotic gradient of 0.3 osmolarity (osM) at pH 2.0 and 4 °C. The relative volume (Vrel = V/Viso, Viso: volume in iso-osmotic solution, volume at time t) was calculated from the Irel − Vrel relationship previously established for PC-liposome model. Spectra are representative of n = 6 and ten replicates for WIV vaccine and split vaccine, respectively, examined at each condition