a, Schematic depicting experimental design to calibrate the FLIRT microscope using a thermochromatic dye. b, Experimental timeline demonstrating the thermochromatic dye upshift and FLIRT experiments to determine chromatic transition point. c, Representative images of transmitted light through the thermochromatic dye after temperature upshift (left column) or after FLIRT using the 16-µm (center column) or 27-µm (right column) diameter masks. N = 4 independent experiments. Scale bar, 10 µm. d,e, Graphs depicting the change in transmitted light intensity with whole coverslip upshift from 8 °C d, or FLIRT targeting using the 16-µm (left) or 27-µm (right) diameter masks while e, the thermochromatic dye-coated coverslip was held at 9, 10, or 11 °C. White dashed circles indicate FLIRT-targeted ROIs. The number of experimental replicates (N) is listed or indicated on individual graphs. Error bars, mean +/− s.e.m.