Two-Photon Excitation Microscopy Protocol

For two-photon excitation microscopy (2PM), we used an FV1200MPE-IX83 inverted microscope (Olympus, Tokyo, Japan) equipped with a 30x/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus) and an FV1200MPE-BX61WI upright microscope equipped with a 25x/1.05 water-immersion objective lens (XLPLN 25XWMP; Olympus) and an InSight DeepSee Laser (Spectra Physics, Santa Clara, CA, USA). The laser power was set to 8–10% and 2–4% for the observation of the intestine and the skin, respectively [33 (link), 38 (link)]. The scan speed was set at 20 μs/pixel. We used 840-nm light to excite CFP. We used an IR-cut filter (BA685RIF-3), two dichroic mirrors (DM505 and DM570), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). Acquired images were analyzed with MetaMorph software (Universal Imaging, West Chester, PA, USA) as described previously [34 (link), 39 (link)].
Confocal images were acquired with an FV1000/IX83 confocal microscope (Olympus) equipped with a 30x/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus).