We here present a method to reconstruct effective free energy landscapes (FELs) of lipid vesicles from the statistical analysis of a large number of microscope images. This method, not only allows us to define possible energy landscapes, but also highlights minority vesicle shapes that were otherwise hidden in the majority. When compared with temporal evolution of deforming lipid vesicles, it was found that the trajectory of deforming vesicles was in accordance with the reconstructed landscape, in which the minority shapes play a key role. When compared with theoretical models, it revealed that the vesicle shapes characterised in the reconstructed FELs were consistent with the theoretically predicted shapes. These results suggest that the FEL analysis can be a useful tool to investigate the morphological dynamics of lipid vesicles, in conjunction with other analytical methods.
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