Iv100 laser scanning microscope

Particles containing Dylight 650 were injected and analyzed by intravital microscopy as described in ref 22 (link). Briefly, experiments were performed using an IV 100 laser scanning microscope (Olympus). Mice (n = 5) were anesthetized with isofluorane, a tail vein catheter was applied, and animals were placed on a 37 °C heated stage in the prone position and kept under anesthesia. The ear was immobilized to an aluminum block with double-sided tape, and vasculature was visualized with a 488 nm laser; the nontumor-bearing ear was used in studies with the LKB498 mice. Mice were then dosed with a bolus injection of Dylight 650-labeled nanoparticles. Fluorescence was measured using a 633 nm laser, and imaging scans were captured every 5 s for 2 h. For circulation analysis, the image files from each scan were exported to ImageJ. Following literature procedures, the images were stacked in groups of four, and fluorescent signal in each stack was analyzed in the region of interest (ROI).^{23 (link),49 (link)} Background corrections were obtained using the initial fluorescence in the ROI before injection. All instrument settings were kept constant for the duration of the study. Particles were dosed at 18.75 mg per kg of body weight.