Spleens or Peyer’s patches of anesthetized mice were imaged. Spleens or Peyer’s patches were surgically exteriorized, immobilized on a microscope stage, and maintained at 37 °C. A Nikon A1 laser scanning confocal microscope with a 20× objective and software NIS-Elements C was used for image acquisition. We used three dichronic mirrors (DM457/514 and DM405/488/561/640), and three bandpass emission filters (482/35, 540/30, 525/50, 595/50 and 700/75). YFP/CFP ratio was obtained by excitation at 458 nm. PE and Alexa-647 were excited at 488 nm and 633 nm, respectively. Images of purified cells in phosphate-buffered saline were also obtained as above. For 2P microscopy, we used a BX61WI/FV1000 upright microscope equipped with a ×25 water-immersion objective lens (XLPLN25XW-MP; Olympus, Tokyo, Japan), which were connected to a Mai Tai DeepSee HP Ti:sapphire Laser (Spectra Physics, Mountain View, CA). The excitation wavelength for CFP was 840 nm. We used an IR-cut filter BA685RIF-3, three dichroic mirrors (DM450, DM505, and DM570), and three emission filters [FF01-425/30 (Semrock) for the second harmonic generation image, BA460-500 (Olympus) for CFP, and BA520-560 (Olympus) for YFP]. Intravital microscopy of mouse calvaria bone tissues was performed using a protocol modified from a previous study; 10–14-week-old mice were anesthetized using isoflurane; the frontoparietal region of the skull bone was exposed, and the internal surfaces of bones adjacent to the bone marrow cavity were observed using multiphoton excitation microscopy. The imaging system was composed of a multiphoton microscope (A1-MP; Nikon) driven by a laser (Chameleon Vision II Ti: Sapphire; Coherent) tuned to 840 nm together with an upright microscope equipped with a 25× water immersion objective (APO, N.A. 1.1; Nikon). We used three dichronic mirrors (DM458, DM506, and DM561) and three bandpass emission filters (417/60, 480/40, and 534/30). Acquired images were analyzed with MetaMorph software (Universal Imaging, West Chester, PA) and Imaris Software (Bitplane AG, Zürich, Switzerland).
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