Two photon microscope

Mice were anesthetized using isoflurane (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). The frontoparietal region of the skull bone was exposed, and the internal surfaces of bones were observed using two‐photon microscopy.10 The imaging system was composed of a Nikon upright two‐photon microscope (A1R‐MP) equipped with a 25× water‐immersion objective (APO, N.A. 1.1; Nikon, Tokyo, Japan) and a Carl Zeiss upright two‐photon microscope (LSM 780 NLO) equipped with a 20× water immersion objective (W Plan‐Apochromat, N.A. 1.0; Carl Zeiss, Oberkochen, Germany). Both systems were driven by a laser (Chameleon Vision II Ti:Sapphire; Coherent, Santa Clara, CA, USA). Intravital bone imaging experiments for TRAP‐tdTomato mice were performed using a Zeiss two‐photon microscope; spectral images were acquired by specialized internal multi‐photomultiplier detectors. Acquired raw images were subjected to spectral unmixing with ZEN software (Carl Zeiss) to create unmixed images that excluded autofluorescence. The excitation wavelength of 940 nm was used. Experiments for a3‐GFP mice were performed using a Nikon two‐photon microscope, and fluorescent images were acquired by external non‐de‐scanned detectors equipped with a bandpass emission filter at 500/50 nm (for GFP). The excitation wavelength was 930 nm.

Channel unmixing was performed on all intravital bone imaging data from Col2.3-ECFP/TRAP-tdTomato mice using a Nikon two-photon microscope. The imaging data of the fluorescence spectra of ECFP, tdTomato, and autofluorescence were obtained using NIS Elements integrated software by manually selecting appropriate pixels on raw images; these spectral libraries were used for channel unmixing algorithms to create unmixed images in which each fluorescence was discriminated and autofluorescence was excluded (Supplementary Figs. 5f–h).

Lycopersicon Esculentum (Tomato) Lectin (LEL, TL) and DyLight 649 (Invitrogen, New York, USA) were injected intravascularly in an attempt to label vascular elements in mice. Image data for tumors and blood vessels were collected by intravital two-photon microscopy (Nikon) on the anesthetized live mouse. Intravital imaging was performed on a Nikon two-photon microscope with a 25×, Apo LWD/1.10 W water immersion objective; a pulsed laser system with a wavelength range of 820-1300/1040 nm was used for excitation of fluorophores. GFP and Cy5/deep-Red were viewed simultaneously with 820 nm excitation, along with 500–550 nm and 601–657 nm emission filters for GFP and Cy5/deep-Red, respectively. During the imaging, the temperature of the mouse was monitored and maintained at 37 °C.