Fig 1.
Temperature dependency of Sirius, mT-Sapphire, and equimolar mixtures of Sirius and mT-Sapphire proteins.
(A) Temperature-dependent fluorescence spectrum of Sirius. The wavelength at 360 ± 10 nm was used for excitation. (B) Temperature-dependent fluorescence spectrum of mT-Sapphire. The wavelength at 400 ± 10 nm was used for excitation. (C) Temperature-dependent fluorescence spectrum of equimolar mixtures of the two FPs. The wavelength at 360 ± 10 nm was used for excitation. (D) Temperature-dependent fluorescence intensity ratio (open red circle and open blue square) of the equimolar mixtures of the two FPs from 5°C to 50°C. The ratio value was plotted against the solution temperature (n = 3). Red and blue lines show the increase in temperature from 5°C to 50°C and the decrease in the opposite direction, respectively. The detailed method for the calculation of the ratio was described in the Methods section. Error bars indicate the standard error (s.e.m.).
Fig 2.
Effects of various ion concentrations on temperature sensing by the equimolar mixtures of Sirius and mT-Sapphire proteins.
(A) Temperature-dependent fluorescence intensity ratio of the equimolar mixtures of the two FPs at various K+ concentrations. (B) Temperature-dependent fluorescence intensity ratio of the equimolar mixtures of the two FPs at various Ca2+ concentrations. (C) Temperature-dependent fluorescence intensity ratio of the equimolar mixtures of the two FPs at various Mg2+ concentrations. (D) pH-dependent fluorescence intensity ratio (509/425 nm) of the equimolar mixtures of the two FPs at various temperature. Cyan; 20°C, yellow-green; 30°C, orange; 40°C. A solution containing 30 mM trisodium citrate and 30 mM borax adjusted to pH 6.0, 7.0, and 8.0 was used. Error bars indicate the s.e.m. (n = 3).
Fig 3.
Monitoring temperature in cells with gTEMP.
(A) Temperature-dependent fluorescence intensity ratio (black circle) of gTEMP expressed in HeLa cell. The ratio in the cytoplasm was plotted against the cellular medium temperature (n = 20). Relative temperature resolution was 0.5°C. (B) Time course of the ratio of gTEMP in a HeLa cell upon IR-laser irradiation. The IR laser was focused in the cytoplasm. We measured and plotted the fluorescence intensity ratio of gTEMP at the focus of the IR laser. The recording rate was 20 fps. The medium temperature was 28°C. (C) The extended figure from (B). Error bars in (A) indicate the s.e.m.
Fig 4.
Monitoring temperature change in mitochondria.
(A) Pseudo-colored ratio image of gTEMP expressed in mitochondria of a HeLa cell upon FCCP stimulation. At time = 0 min, 10 μM FCCP was added to the cell. (B) Time course of the ratio in mitochondria with FCCP and without FCCP. The temperature scale (5°C) in Fig 4B was estimated from the slope value (0.031 ratio/°C) of S5 Fig. The medium temperature was 37°C. Scale bars indicate 20 μm (A).
Fig 5.
Monitoring temperature difference in cells.
(A) Pseudo-colored ratio image of gTEMP ubiquitously expressed in a HeLa cell. (B) Plot of gTEMP ratio in cytoplasm and nucleus regions in each HeLa cell (n = 13). (C) Histogram of the gTEMP ratio difference between cytoplasm and nucleus regions converted from (B). The average temperature difference in Fig 5C was 2.9 ± 0.3°C estimated from the slope value (0.045 ratio/°C) of S6 Fig. The medium temperature was 37°C. Scale bar indicates 20 μm (A).
Fig 6.
Monitoring temperature in medaka embryo.
The bright field image (upper) and pseudo-colored ratio image (bottom) of gTEMP expressed in a medaka embryo. The medium temperature was 25°C. Scale bars indicate 250 μm.