Xlumplfln 20 water immersion objective

In vivo microscopy was performed using an Olympus FV1000 multiphoton/confocal imaging system following procedures described previously 47 ,55 (link). Animals were used with Institutional Subcommittee on Research Animal Care guidelines. 5 × 10⁵ MC38-H2B-mApple cells in 50 μL PBS were injected under the fascia two days after DSWC implantation surgery and imaged ~7 days later upon tumor formation. MN patches were applied onto the tissue within the window chamber, and glass coverslips were replaced over the patch to protect the tissue. 24 h later, patches were removed, and tissue was imaged after adding saline and replacing the coverslip. An XLFluor X2 air objective (numerical aperture 0.14, Olympus) was used for low-magnification imaging of the entire tumor and MN delivery region. In contrast, higher-magnification imaging was conducted with an XLUMPLFLN ×20 water immersion objective (numerical aperture, 1.0, Olympus). Sequentially scanned images were acquired with 559- and 633-nm diode lasers and a DM405/488/559/635-nm dichroic beam splitter 42 (link),50 (link). Empty patches lacking both CpG and dye were re-applied under coverslips for 24 h repeated twice to mirror the serial treatment scheme in the subcutaneous experiments. Longitudinal imaging was performed for up to 9 days post-treatment.

In HT1080 xenograft experiments, NP was injected via tail-vein catheter immediately after mixing to a final 1 × PBS solution. Intravital microscopy was performed on an Olympus FV1000 Confocal-Multiphoton Imaging System using a XLUMPLFLN × 20 water-immersion objective (NA 1.0; Olympus America); 2 × digital zoom; sequential scanning using 405-nm, 473-nm, 559-nm and 635-nm diode lasers and a DM405/473/559/635-nm dichroic beam splitter; and collection of emitted light using beam splitters (SDM473, SDM560 and/or SDM 640) and emission filters BA430-455, BA490-540, BA575-620 and BA655-755 (all Olympus America). Dorsal window chamber imaging was performed following previously described procedures61 (link), such that two million HT1080-53BP1-mApple cells were suspended in 50 μl PBS, injected under the fascia of nu/nu mice (Cox7; MGH) 30 min after surgical chamber implantation and imaged 2 weeks later. Terminal orthotopic imaging was performed by surgically opening the peritoneal cavity 24 h following intraperitoneal drug injection.

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.