Quantitative Fluorescence Imaging of Tumor Vasculature

Fluorescence analyses were performed using an Olympus MVX10 microscope with a 2X objective (NA, 0.5) and an optical zoom of 0.63-6.3x. Excitation and emission filters were 470 ± 40 nm and 525 ± 50 nm for eGFP, 560 ± 55 nm and 645 ± 75 nm for Texas Red dextran, and 740 ± 35 nm and 780 longpass filter for near-infrared indocyanine green. Exposure time varied from 300-500 ms for initial scans of whole tissue sections to 15 ms for quantitative analysis of tumor regions. For Texas Red dextran, total fluorescence intensity in a region of interest was converted to sum voxel intensity/g tissue. Volume was calculated as area (cm²) × 0.002 cm thickness corrected for density 1.04 g/cm³. To convert fluorescence intensity to concentration, standard curves were generated (Supplementary Fig. 3), similar to autoradiography (46 (link)). Brain (500 mg) was excised and homogenized to uniformity with 100 μL of saline containing different concentrations of Texas Red dextran. The final mixture was flash frozen in isopentane and sliced into 20 μm sections. Similarly, blood samples were spiked with concentrations of dye, 1 μL samples were placed on glass slides and dried, and then total fluorescence intensity for the blood drop was measured. Texas Red dextran fluorescence intensity did not differ between standards prepared from brain or tumor, or from solutions of differing pH (6.0 −7.6) or Na⁺/Ca²⁺ concentration (data not shown). Texas Red dextran sum intensity was stable within ± 5% with repeat fluorescent exposures (15 ms – 1500 ms). Fluorescent image analysis was performed using Slidebook 5.0 program (Olympus). Vascular density and surface area were calculated using binary masks where vessels were defined by indocyanine green fluorescence ≥3 fold above background.