Abstract: Cell migration, formation of cellular protrusions (e.g. blebs, filopodia), and structural reorganization are important phenomena in cell biology. Precise quantifications of movement/deformation are crucial to understand these processe at different levels of organization. We apply computer vision methods for combined optical flow (OF) and muti‐scale (MS) motion estimation of membrane translations, end growing and protrusion formation in fluorescence microscopy images. For these cases we bound OF error and optimal sampling rate, in order to guide biologists on their experimental conditions. We also show the advantages of OF methods compared with manual segmentation and tracking.