Fig. 3.

Clinical NIR imaging to quantify lymphatic flow in the upper extremity. A custom NIR imaging system (FD-1665; FluxData Inc., Rochester, NY, USA) was used to assess lymphatic contraction frequency in a healthy human subject after ICG injection in the second, third, and fourth web spaces of both hands as described in ClinicalTrials.gov NCT2680067. NIR excitation (<1.8 mW/cm²) was monitored with a Thorlabs PM16-121 power meter adjacent to the first web space. After injections, the upper extremities were imaged for 10 minutes to observe lymphatic flow. Visible and NIR (>800 nm) images were collected simultaneously; ICG fluorescence images were used to pseudocolor the visible image to provide anatomic localization (a). The ROI was positioned over lymphatics to calculate the mean pixel value for each NIR frame; peaks associated with lymphatic contractions were counted to calculate cpm. Representative images of the left hand (b) obtained from real-time video (Additional file 1: Movie 1) and right antecubital fossa (c) (Additional file 2: Movie 2) with the ROI and the respective cpm (green)