Intravital Microscopy of Tumor Vasculature

TNPs were injected at the indicated dose (1 mg kg⁻¹ unless otherwise stated) via tail-vein catheter immediately after mixing to a final 1 × PBS solution, at a final volume of 100 μl. Intravital microscopy was performed on an Olympus FV1000 multiphoton imaging system using a XLUMPLFLN × 20 water immersion objective (numerical aperture=1.0; Olympus America). Images were scanned sequentially using 405-, 473-, 559- and 633-nm diode lasers in combination with a DM405/488/559/635-nm dichroic beam splitter. Emitted light was then separated and collected using appropriate combinations of beam splitters (SDM473, SDM560 and/or SDM 640) and emission filters BA490–540, BA575–620, BA575–675 and/or BA655–755 (all Olympus America). Dextran pacific blue (λ_ex=405 nm) was injected to initially image TMV as previously described29 (link). Briefly, 500-kDa amino-dextran (Thermo) was labelled with Pacific Blue succinimidyl ester (Thermo), purified using 30 kDa MWCO centrifugal filtration (Amicon), and 250 μg i.v. injected 10 min before imaging. Dorsal window chamber imaging was performed following previously described procedures29 (link)69 70 (link). Briefly, 2 million HT1080–53BP1-mApple cells were suspended in 50 μl PBS and injected under the fascia of nu/nu mice (Cox7, MGH) 30 min following surgical chamber implantation, and imaged 2 weeks later.