Dataset of stimulated emission depletion (STED) microscopy images in living cells. Additionally, wide field epifluorescence time lapses were performed to investigate cell proliferation. Various human cell lines (U2OS, HeLa, and RPE-1) and structures (nuclear pore complex, Golgi, mitochondria and actin) were imaged. The dataset contains both long-term (20 h) studies on cell proliferation and survival after high-resolution (50 nm) STED imaging, and the short-term stress response of cells by observing the cytosolic calcium levels during various types of imaging.