Rhodamine b solution

On day 14, one randomly selected demineralized block per group was sectioned into 3–5 slices (thickness, 300 μm) using a Kavo sander under running water. The prepared enamel slices were placed in rhodamine B solution (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Item No. 02558) under protection from light, and placed in a constant temperature incubator at 37°C for 48 h for staining. Subsequently, deionized water was used to rinse the blocks three times (5 min each) to wash away the dye on the surface. The blocks were dried and placed on slides, sealed with glycerol, and viewed under a laser scanning confocal microscope (Leica Measuring Instruments (China) Co., Ltd., Model: TCS-SP8) to observe the fluorescence penetration in the demineralized areas. Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) software was used to analyze the enamel fluorescence area (Area, μm²), total fluorescence in the measured area (IOD), and mean fluorescence in the measured area (MIOD) values using the following formula: MIOD = IOD/Area

To prepare it for fluorescence imaging, silk scaffold was polymerized in a well with glass bottom compatible with high resolution imaging. The scaffold was then incubated for 30 min in 0.5% w/w Rhodamine B solution (Sigma Aldrich, 83689) in PBS and rinsed thoroughly to remove the excess dye. On separate samples, in order to visualize live cell distribution on the silk scaffold 10 days after cell seeding, cell-laden scaffolds were incubated with 2 µM Calcein AM (ThermoFisher Scientific, C3100MP) for 30 min at 37°C and then rinsed thoroughly with cell culture medium. Imaging was performed on a Nikon A1RHD confocal microscope equipped with a 20x objective. Image processing was performed in NIS-elements software (Nikon).

To measure the spatial temperature profile inside the cell incubation chamber, as well as the temperature increase inside nuclei during temperature stimulation experiments, we used temperature-sensitive decrease in quantum efficiency that has been well described for certain dyes (usually in the red spectrum) before (Hirsch et al., 2018 (link); Mittasch et al., 2018 (link); Singhal and Shaham, 2017 (link)). For chamber measurements, rhodamine B solution (Sigma, 02558) was diluted to 10% in water and image stacks were acquired in the red channel during laser application. For nuclear measurements, mCherry-H2b transfected cells were recorded. Both dyes were calibrated by precise changing of the bulk temperature of the incubation chamber (Figure 1—figure supplement 1). For rhodamine, thermophoretic effects (lower dye intensity due to concentration difference, not quantum yield) were determined to correct measurements.