In Vivo Multiphoton Imaging of Tumor Microenvironment

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.