Excitation 472 30

Live-cell imaging was performed on an epi-fluorescence microscope (Leica DMI6000B) equipped with an adaptive focus system and an on stage incubator chamber (Tokai Hit GM-8000) to maintain the temperature at 37 °C and 5% CO₂ during the imaging period. Images were acquired using an oil-immersion 100× objective (Leica, HCX PL APL, n.a. 1.4) and an light-emitting diode (LED) light source (Lumencor Sola, Beaverton, OR). CRY2 was activated by either a single blue light pulse of 2 s or an intermittent blue light pulse of 200 ms at every 5 s. The LED intensity can be adjusted to a range between 1.2 × 10³ mW/cm² and 9.7 × 10³ mW/cm². Unless otherwise specified, the intensity of the blue light pulse used to activate CRY2 was 9.7 × 10³ mW/cm². Additionally, low blue light intensity (7.9 mW/cm²) was produced by filtering the brightfield light source of the microscope through a blue bandpass filter (Chroma, 460/20). The GFP fluorescence signal was detected using a commercial GFP filter cube (Leica, excitation 472/30, dichroic mirror 495, emission 520/35). mCherry was excited using green light (~550 nm, 9.7 × 10³ mW/cm²), and the mCherry fluorescence signal was detected using a commercial Texas Red filter cube (Leica, excitation 560/40, dichroic mirror 595, emission 645/75). Movie frames were collected every 5 s at 200 ms exposure.