In Vivo Multimodal Intravital Imaging

Mice were imaged as previously described^{31 (link)}. In short, mice were sedated and placed face-up in a custom-designed imaging box. Time-lapse images of the entire tumor volume were acquired for a maximum of 13 hours. The minimal time interval between serial images was set to 45 minutes. For tile-scans, images of the complete z-stack of the tumor were acquired to a depth of 300 μm, with a step size of 3 µm (typically 70–100 images in each z-stack). In a group of 4 mice, blood vessels were imaged by intravenous injection of 70 kDa Dextran–Texas Red (Invitrogen Life Technologies) or 2.000.000 kDa Dextran-Rhodamine (Thermofisher scientific).
Imaging was performed on an inverted Leica SP8 multiphoton microscope with a chameleon Vision-S (Coherent Inc., Santa Clare, CA, www.coherent.com), equipped with a 25 x water objective (HCX IRAPO NA0.95 WD 2.5 mm) with four HyD detectors: HyD1 (560–650 nm), HyD2 (500–550 nm), HyD3 (455–490 nm), and HyD4 (<455 nm). Images were acquired at a wavelength of 960 nm, where H2B Dendra2 as well as both Dextran reagents showed sufficient signal intensity. Collagen (second harmonic generation) was detected using HyD4, H2B-Dendra2 was detected with HyD2 and Dextran reagents were detected with HyD1. Scanning was performed in a bidirectional mode at 400 Hz and 12 bit, with a zoom of 1 × , 512 × 512 pixels.