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Increasing Indocyanine Green Fluorescence With Organic Solvents For Experimental Lymphangiography
Jason Gardenier, MD, Sameer Masand, BS, Geoffrey Hespe, BS, Ira Savetsky, MD, Gabriela Garcia Nores, MD, Jessie Yu, MD, Raghu Kataru, PhD, Babak Mehrara, MD.
Memorial Sloan Kettering Cancer Center, New York, NY, USA.

PURPOSE:
Indocyanine green (ICG) is a widely-used near-infrared (NIR) fluorescent contrast agent used in in-vivo lymphangiography. However, the use of ICG is limited by its intensity and ability to visualize deep lymphatics. Previous reports have suggested that organic solvents can increase the fluorescent intensity of ICG. Therefore the purpose of this study was to optimize the concentration of ICG and determine if amounts of ethanol added to the solvent can increase the fluorescent intensity of ICG and improve lymphatic visualization.
METHODS:
We first determined the optimal concentration of ICG in deionized water using a spectrophotomator and monochromator to measure the fluorescent intensity of a dilution series of ICG. We then prepared a series of ICG solutions with various proportions of ethanol and analyzed them with a fluorescent microscope with an excitation wavelength of 770nm and an emission wavelength of 800nm—quantifying fluorescent intensity with metamorph software. We then performed lymphangiography in mouse hindlimbs using ICG solutions containing 25%, 10%, and 0% ethanol.
RESULTS:
We found that the optimal concentration of ICG for fluorescent intensity was 0.05mg/ml, which provided a 4-fold increase in fluorescent intensity over previously-published concentrations. Increasing ethanol concentrations increased fluorescent intensity with 10% ethanol doubling fluorescent intensity and 25% ethanol tripling fluorescent intensity without apparent toxicity locally.
CONCLUSION:
We determined the optimal concentration of ICG for NIR lymphatic imaging and provided a cost-effective way of markedly improving the quality of lymphangiography using the organic solvent ethanol. Future studies will analyze cytotoxicity and application to experimental and clinical lymphangiography.


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