Temperature measurement for rat neonatal cardiomyocytes. (A) Fluorescence images of Fluo4 (top), EuTTA (middle), and Rh101 (bottom) of cardiomyocytes on a RT nanosheet. Images were averaged from 600 frames (for 18 s) during observation. (B) Time course of changes in the F.I. of Fluo4 (top), EuTTA (middle), and Rh101 (bottom). Data obtained from cells 1–5 (see A, top; Video 6, and Video 7). (C) Top: Time course of changes in average Fluo4 F.I. in cardiomyocytes on RT nanosheets. Number of cells, 75 (n = 17). Bottom: Time course of changes in the F.I. of EuTTA (red) and Rh101 (blue) on RT nanosheets cultured with cardiomyocytes. Different cells were used in top and bottom, and Fluo4 was not loaded in cells in bottom. The photobleaching effects were corrected. Number of cells, 142 (n = 36). See Materials and methods for details. (D) Superimposed data of Fluo4 (top), and EuTTA (red), Rh101 (blue), and EuTTA/Rh101 (black; bottom). Data obtained by 10 stimuli were superimposed. A calibration bar of 0.01°C was calculated from the temperature sensitivity of the EuTTA/Rh101 ratio (i.e., −3.35%/°C; cf. Fig. 1 D). Arrows in B, C, and D indicate the points at which electrical stimuli were applied. a.u., arbitrary units.