A Spy1000 system (Novadaq Technologies Inc Mississauga, Ontario, Canada) was used. This system consists of an 806nm laser providing maximum illumination intensity approximately 30 mW/cm2 at a fixed focal length. This instrument was modified to provide continuous operation and adjustment of laser intensity, and was equipped with a second PAL camera. Filters permitted the measurement of light above 815nm. The video outputs of the camera were attached to the network using an Axis 241SA video server (Axis, Sweden, Lund) that was monitored using SecuritySpy (Ben Bird, www.bensoftware.com ) that converted motion JPEG image streams to QuickTime movies (Apple Computer Inc). Individual JPEG image sequences were then exported and read for further analysis with ImageJ.
ICG (Acorn or Pulsion) was dissolved in the distilled water at 0.1 μg/μl and 10 μl (1 μg) of ICG solution was intra-dermally injected into the footpad using a 30G needle. Before ICG injection, fur was removed from legs with hair removal lotion. Animals were placed in recumbent position and legs were restrained with tape on an isothermal gel pad during imaging sessions. ICG fluorescence was recorded for 1–2 hours immediately and again 24 hours for 5 min after the ICG injection. The QuickTime movies were examined and the time for ICG appearance in popliteal lymph node (PLNs) after the footpad injection was recorded. Sequential images from the movie file were exported and the intensity of ICG fluorescence of PLNs and footpads were determined using ImageJ. A fixed circular region of interest (ROI) was drawn over the vessels or PLNs and nearby background tissue. The signal intensity was defined as Signal intensity – background intensity. The following outcome measures were derived from the ICG-NIR images: 1) T-initial (T-in), which is the time that it takes for the ICG to be detected in vessels; 2) S-max, which is the maximum ICG signal intensity observed in the PLN during the first day imaging session; 3) T-max, which is the time it takes for a PLN to achieve maximal ICG signal intensity; 4) % Clearance, which is an assessment of ICG wash out through the lymphatics and is quantified as the percent difference of ICG signal intensity between the two ICG-NIR images from the ROI of the PLNs or footpad at a) S-max (first day) and b) 24 hours post ICG injection; and 5) pulse, which is the ICG pulses that pass the ROI within 400 seconds.
ICG (Acorn or Pulsion) was dissolved in the distilled water at 0.1 μg/μl and 10 μl (1 μg) of ICG solution was intra-dermally injected into the footpad using a 30G needle. Before ICG injection, fur was removed from legs with hair removal lotion. Animals were placed in recumbent position and legs were restrained with tape on an isothermal gel pad during imaging sessions. ICG fluorescence was recorded for 1–2 hours immediately and again 24 hours for 5 min after the ICG injection. The QuickTime movies were examined and the time for ICG appearance in popliteal lymph node (PLNs) after the footpad injection was recorded. Sequential images from the movie file were exported and the intensity of ICG fluorescence of PLNs and footpads were determined using ImageJ. A fixed circular region of interest (ROI) was drawn over the vessels or PLNs and nearby background tissue. The signal intensity was defined as Signal intensity – background intensity. The following outcome measures were derived from the ICG-NIR images: 1) T-initial (T-in), which is the time that it takes for the ICG to be detected in vessels; 2) S-max, which is the maximum ICG signal intensity observed in the PLN during the first day imaging session; 3) T-max, which is the time it takes for a PLN to achieve maximal ICG signal intensity; 4) % Clearance, which is an assessment of ICG wash out through the lymphatics and is quantified as the percent difference of ICG signal intensity between the two ICG-NIR images from the ROI of the PLNs or footpad at a) S-max (first day) and b) 24 hours post ICG injection; and 5) pulse, which is the ICG pulses that pass the ROI within 400 seconds.
