Ba460 500

For long-term organ-scale imaging, we partially cut off the capsule adjacent to the apex tip of cochlear duct using tweezers carefully and the semicircular canals were removed. The isolated cochlea was put onto the dish as described above. For microscopy, we used an incubator-integrated multiphoton fluorescence microscope system (LCV-MPE, Olympus) with a × 25 water-immersion lens (NA = 1.05, WD = 2 mm, XLPLN25XWMP2, Olympus). The excitation wavelengths were set to 840 nm for CFP (InSight DeepSee, Spectra-Physics). Imaging conditions for the FRET biosensor were as follows: scan size: 800 × 800 pixels; scan speed: 10 μs/pixel; IR cut filter: RDM690 (Olympus); dichroic mirrors: DM505 and DM570 (Olympus); and emission filters: BA460-500 for CFP and BA520-560 for FRET detection (Olympus).

The establishment of transgenic mice expressing AKAR3EV (PKAchu mice) was described previously (Kamioka et al., 2012 (link)). Briefly, 8- to 13-week-old female mice were used for the in vivo imaging. The ear hair was removed with a razor 1 d before the experiments. Mice were anesthetized with 1.5% isoflurane (FUJIFILM Wako Pure Chemical Corp.) inhalation and placed in a side-lying position on an electric heater maintained at 37°C. The ear skin was placed on the cover glass. Two-photon excitation microscopy was performed with an FV1200MPE-IX83 inverted microscope (Olympus) equipped with a ×30/1.05 silicon oil-immersion objective lens (XLPLN 25XWMP; Olympus), an InSight DeepSee Ultrafast laser (Spectra Physics), an IR-cut filter (BA685RIF-3), two dichroic mirrors DM505 (Olympus), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). The excitation wavelength was 840 nm.
The animal protocols were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (approval no. 22063).

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.