Xlumplfln 20x water immersion objective

Intravital microscopy was performed on an Olympus FV1000 confocal-multiphoton imaging system using a XLUMPLFLN 20x water immersion objective (NA 1.0; Olympus America) with 2x digital zoom. Images were scanned sequentially using 405-nm, 473-nm, 559-nm, and 635-nm diode lasers with a DM405/473/559/635-nm dichroic beam splitter; emitted light was collected using combinations of 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 procedures (16 (link)); briefly, 2 million HT1080-membrane-mApple cells in 50 μl PBS were injected under the fascia of nu/nu mice (Cox7, MGH) 30 min after surgical chamber implantation and imaged two weeks later.

Confocal microscopy was performed with a FluoView FV1000 multi-photon confocal imaging system (Olympus). For confocal microscopy imaging of excised and cleared tissues, an XLFluor 2x air objective (NA=0.14, Olympus) was used for low-magnification imaging, while an XLUMPLFLN 20x water immersion objective (NA=1.0, Olympus) was used for high-magnification imaging. Four fluorescent channels were imaged. DAPI, GFP/A488, A555/Rhodamine/BODIPY-TMR, A647/VT680/SiR signals were excited sequentially with 405, 473, 559, and 635 nm diode lasers, respectively, in combination with DM405, 473, 559, and 635 nm dichroic beam splitters. The emitted light was separated with SDM473/560/640 nm beam splitters. DAPI, GFP/A488, A555/Rhodamine/BODIPY-TMR, A647/VT680/SiR signals were detected with BA430-455, BA490-540, BA575-620, and BA655-755 nm emission filters, respectively. Each fluorescence channel was imaged sequentially using distinct excitation and emission filter sets to ensure minimal bleed-through between channels. All lasers, beam splitters, and emission filters were purchased from Olympus. Images were collected at 150 μm or 10 μm interval in z-direction for low or high-magnification imaging, respectively.

TNPs were injected at the indicated dose (1 mg kg⁻¹ unless otherwise stated) via tail-vein catheter immediately after mixing to a final 1x phosphate-buffered saline (PBS) solution, at a final volume of 100 μl. Intravital microscopy was performed on an Olympus FV1000 multiphoton imaging system using a XLUMPLFLN 20x water immersion objective (NA 1.0; Olympus America). Images were scanned sequentially using 405-nm, 473-nm, 559-nm 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 described^{28 (link)}. Briefly, 500 kDa amino-dextran (Thermo) was labeled with Pacific Blue succinimidyl ester (Thermo), purified using 30 kDa MWCO centrifugal filtration (Amicon), and 250 μg intravenously injected 10 min prior to imaging. Dorsal window chamber imaging was performed following previously described procedures^{28 (link),67 ,68 (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 two weeks later.