Supplementary Figure 3: mCherry fluorescence-based temperature and gradient calibration.

a, Schematics and representative images of FLIRT microscope thermal calibration using mCherry::HistoneH2B fluorescence in late-stage embryos (~100–200 cells) and either whole-embryo upshift (left) or while FLIRT targeting with increasing infrared laser power using the 16-µm or 27-µm diameter masks (right). N = 7 biologically independent embryos. b, Quantification of mCherry fluorescence intensity levels³⁷ on temperature upshift (left) or using FLIRT with increasing infrared laser power (right). Error bars, mean +/− s.d. c, Schematic of FLIRT thermal gradient measurement across an embryo. d, Characterization of FLIRT thermal gradient. A gradient of ~4–8 °C was maintained on the other end of the same embryo, depending on the infrared mask used and hold temperature set point. Dashed lines represent the mask edges; error bars, mean +/− s.e.m. e, Measurement of the effect of hold temperature on FLIRT thermal gradient when FLIRT is used to heat the mask region to ~25.5 °C with varying hold temperatures (10–16 °C) and laser powers (8.5–13.9 mW, right table). The green dashed line at x = 16 µm shows the mask edge and the black dashed line at y = 24.5 °C shows the restrictive temperature for myosin-II(ts) mutants (Fig. 1c,d and Supplementary Figs. 4 and 6a). Error bars, mean +/− s.e.m. Note: 26 °C temperature upshift and 16 °C hold data are also shown in both d and e. f, Bar graph showing cell division phenotypes for control embryos at different hold temperatures.