Temperature measurement for HeLa cells. (A) Fluorescence images of Fluo4 (top) in HeLa cells and EuTTA (bottom) of a RT nanosheet. Left and right: Before and after induction of Ca²⁺ increases by 2 µM ionomycin, respectively. See Video 4. (B) Time course of changes in the F.I. of Fluo4 in HeLa cells on RT nanosheets before and after application of ionomycin (red line) or DMSO (black line). (C) Time course of the EuTTA/Rh101 ratio of RT nanosheets with cells. F.I. corrected by that for the medium. See Fig. S9. In B and C: number of cells, 16 (n = 3) and 10 (n = 2) for ionomycin and DMSO, respectively. Data shown as mean ± SEM. The arrow indicates the point at which ionomycin or DMSO was applied. (D) Fluorescence images of Fluo4 (top) and ER thermo yellow (bottom) in HeLa cells on an EuTTA nanosheet. Left and right: Before and after induction of Ca²⁺ increases by 2 µM ionomycin, respectively. See Video 5. (E) Time course of changes in Fluo4 F.I. in HeLa cells on EuTTA nanosheets before and after application of ionomycin (red line) or DMSO (black line). (F) Time course of changes in the F.I. of ER thermo yellow in HeLa cells on EuTTA nanosheets. In E and F: number of cells, 12 (n = 3) and 8 (n = 2) for ionomycin and DMSO, respectively. Data shown as mean ± SEM. The arrow indicates the point at which ionomycin or DMSO was applied. Calibration bars in C and F were calculated from the temperature sensitivity of RT nanosheets (−3.35%/°C, cf. Fig. 1 D) and ER thermo yellow (−3.9%/°C; cf. Arai et al., 2014), respectively. Fluorescence images in A and D are averaged from 30 frames before (0–58 s) and after (180–238 s) the ionomycin treatment. a.u., arbitrary units.