Heat stress responses are analyzed in cancer cells by applying different microscopy techniques for targeting various fluorescently labeled or native structures. Thermotreatments are performed at 40, 45, 50, and 56 °C, respectively, for 30 min each, while controls were kept at 37 °C. Actin cytoskeletons labeled with Alexa Fluor® 488-conjugated phalloidin are imaged by wide-field fluorescence microscopy (WFFM). Structural plasma membrane stabilities are labeled with fluorescent quantum dots and analyzed by laser scanning microscopy (LSM). High-resolution atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to study morphological features and surface structures. Fluorescence images reveal F-actin to be a comparatively thermolabile cell component showing distinctive alteration after heat treatment at 40 °C. Destabilization of actin cytoskeletons proceed with increasing stress temperatures. Active reorganization of plasma membranes coincidental to heat-induced shrinkage and rounding of cell shapes, and loosening of monolayered tissue are observed after treatment at 45 or 50 °C. Active stress response is inhibited by stress at 56 °C, because actin cytoskeletons as well as plasma membranes are destroyed, resulting in necrotic cell phenotypes. Comparing data measured with the same microscopic technique and comparing the different datasets with each other reveal that heat stress response in MX1 cells results from the overlap of different heat-induced subcellular defects.