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Two-photon excitation microscopy was performed with FV1200MPE-BX61WI upright microscopes, equipped with a 25X/1.05 water-immersion objective lens (XLPLN 25XWMP; Olympus, Tokyo, Japan) and an InSight DeepSee Ultrafast laser (0.95 W at 900 nm) (Spectra-Physics, Santa Clara, CA, USA). The laser power used for observation was 2–4% for mice expressing EKAREV-NES/NLS, 10–15% for Fucci mice. Scan speed was 8 μs/pixel. Images were recorded every 5 min for long-time imaging or every 1.5 min or 2.5 min for short-time imaging of Eisuke mice expressing EKAREV-NLS, and every 1 hr for imaging of Fucci mice and Eisuke mice expressing EKAREV-NES. The excitation wavelength was 840 nm for mice expressing a FRET biosensor and 910 nm for Fucci mice. For FRET mouse imaging, we used an IR-cut filter, RDM690 (Olympus), two dichroic mirrors, DM505 (Olympus) and DM570 (Olympus), and three emission filters, FF01-425/30 (Semrock, Rochester, NY) for second harmonic generation imaging, BA460-500 (Olympus) for CFP, and BA520-560 (Olympus) for FRET. For Fucci mouse imaging, we used an IR-cut filter, RDM690 (Olympus), two dichroic mirrors, DM505 (Olympus) and DM570 (Olympus), and three emission filters, FF01-472/30 (Semrock) for second harmonic generation images, BA495-540 (Olympus) for mAG, and BA575-630 (Olympus) for mKO2. Images were acquired with a viewfield of 0.213 mm² in 2–3 μm steps.

We used an FV1000MPE‐BX61WI upright microscope (Olympus, Tokyo, Japan) equipped with a 25×/1.05 water‐immersion objective lens (XLPLN 25XWMP; Olympus), and an InSight DeepSee Ultrafast laser (0.95 W at 900 nm; Spectra Physics, Mountain View, CA). The excitation wavelength for cyan fluorescent protein (CFP) was 840 nm. An IR‐cut filter, BA685RIF‐3, two dichroic mirrors, DM505 and DM570, and four emission filters, FF01‐425/30 (Semrock, Rochester, NY) for the second harmonic generation (SHG), BA460‐500 (Olympus) for CFP, BA520‐560 (Olympus) for yellow fluorescent protein (YFP), and 645/60 (Chroma Technology Corp., Bellows Falls, VT) for Qtracker 655 (Life Technologies, Carlsbad, CA), were used. The microscope was equipped with a two‐channel GaAsP detector unit and two built‐in photomultiplier tubes. FluoView software (Olympus) was used to control the microscope and to acquire images, which were saved in the multilayer 16‐bit Tagged Image File Format. Recorded images were processed and analyzed with MetaMorph software (Molecular Devices, Sunnyvale, CA).