Measuring Intracellular Temperature Profiles

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.