Fluorescence characteristics of RT nanosheets. (A) Illustration showing preparation of a RT nanosheet; viz., chloroform (volume of 100 µl), containing the temperature-sensitive fluorescent dye EuTTA, the temperature insensitive fluorescent dye Rh101 and PMMA, is spin-coated on a coverslip of a culture dish. (B) Fluorescence images of EuTTA (left) and Rh101 (right) of a RT nanosheet. F.I. is expressed in arbitrary units (a.u.). (C) Temperature dependence of the F.I. of EuTTA (red) and Rh101 (blue). F.I. normalized at 36°C for both EuTTA and Rh101. Slopes, −3.42 and −0.06%/°C for EuTTA and Rh101, respectively. n = 3. See Fig. S2. (D) Temperature-dependence of the EuTTA/Rh101 ratio. Slope, −3.35%/°C. (E) F.I. of EuTTA (red) and Rh101 (blue) at various pH levels. Note that red circles overlap with blue circles. F.I. normalized at pH 7. n = 3. (F) EuTTA/Rh101 ratio at various pH levels. (G) F.I. of EuTTA (red) and Rh101 (blue) at various ionic strengths. F.I. normalized at 150 mM. n = 3. See Fig. S4. (H) EuTTA/Rh101 ratio at various ionic strengths. In E–H, error bars (SEM) are within plots. (I) Illustration showing a microscopic system for optical heating of a RT nanosheet. Focused IR laser (λ = 1,475 nm; 8 mW) increased temperature around the laser spot. DM, dichroic mirror; DM1, custom-made DM (Sigma Koki); DM2, FF409-Di03 (Semrock); DM3, YOKO-T405/488/561 (Semrock). The optical paths are simplified. See Materials and methods for details. (J) Time-course of changes in the F.I. of an RT nanosheet upon an increase in temperature by IR laser irradiation for 0.5 s (as indicated by “Heating”). F.I. around the laser spot (393 µm2) was measured. F.I. was normalized by that obtained before heating. The photobleaching effects were corrected. (K) Enlarged graphs of J showing the onset (left) and end (right) of heating.
