For intravital microscopy, mice were anesthetized with isofluorane and stably mounted onto a temperature-controlled platform (37°C). FBR elicited by an implanted mPCL-CaP scaffold was monitored using a custom intravital multiphoton microscope (LaVision BioTech)31 (link) with three Ti:Sapphire lasers (Chameleon-XR, Coherent) and two Optical Parametric Oscillators (APE/Coherent), resulting in a tuneable excitation range from 800 to 1300 nm (Fig S1 ). Multi-spectral detection was performed using up to 5 backward or 2 forward photomultipliers (PMTs) using up to three excitation wavelengths in two consecutive scans, to separate the following excitation and emission channels: GFP (920 nm; 525/50 nm); Hoechst 33342 (920 nm; 450/60nm), Rhodamine (1090 nm; 595/40 nm), SHG (1090 nm; 525/50 nm), THG (1180 nm; 387/15 nm) and AlexaFluor750 (1180 nm; 810/90 nm).
For intravital detection, long-working distance 16x NA 0.8 water or 25x NA 1.05 multi-immersion oil/water objectives (Olympus) were used. Sequential 3D stacks were obtained with 5–10 μm step-size reaching up to 200 μm penetration depth. Images were acquired in a random fashion within the subcutaneous tissue up to the dermis. Perfused blood vessels were visualized by i.v. injection of Rhodamine- or AlexaFluor750-conjugated dextran (70 kD; Invitrogen; 1 mg/mouse). mPCL-CaP scaffolds, in vitro, were analyzed using SHG and THG imaging.
For intravital detection, long-working distance 16x NA 0.8 water or 25x NA 1.05 multi-immersion oil/water objectives (Olympus) were used. Sequential 3D stacks were obtained with 5–10 μm step-size reaching up to 200 μm penetration depth. Images were acquired in a random fashion within the subcutaneous tissue up to the dermis. Perfused blood vessels were visualized by i.v. injection of Rhodamine- or AlexaFluor750-conjugated dextran (70 kD; Invitrogen; 1 mg/mouse). mPCL-CaP scaffolds, in vitro, were analyzed using SHG and THG imaging.