Dm570

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.

See Fig. 2b,c and Supplementary Table 1b,c. The following excitation filters, dichroic mirrors and emission filters combined with ND filters were used:
Exciter: 550.0 IF 10 (550 ± 5 nm) (Cheshire Optical)
Dichroic mirror: DM570 (Olympus)
The excitation light density above the objective was 3.4 W cm⁻².
Emitter: BA575IF (575 nm < ) (Olympus) combined with NDX006 (6% transmittance) (Asahi Spectra)