Introduction: Intra-operative identification of tumors can be challenging for surgeons when performing transoral robotic surgery (TORS) for oropharyngeal carcinoma. The target tumor moves and deforms, becomes occluded by blood during surgery, and can be difficult to differentiate from surrounding tissues. We aim to develop and evaluate small biocompatible near infrared fluorescent (NIRF) markers to mark the margin of the target tumor for guiding TORS.
Methods: Each marker is comprised of a solution of clinically approved Indocyanine green (ICG), Dermabond and acetone. A light source then excites the marker, which fluoresces at a specific wavelength. By using near infrared (NIR) camera, we can specifically observe the excited fluorescent light from the markers. The surgeon manually marks the margin of the tumor with four submucosal injections of NIRF solution. When the solution contacts tissue, it quickly hardens into a bead, maintains its position at the subdermal injection site, and the NIR signal penetrates the tissue. We have designed and built a camera stand that protects the samples from external light to achieve constant lighting and imaging conditions (Fig. 1) and two studies have been performed.
Results: The first study was designed to test if the markers maintain their signal to noise ratio (SNR) intensity over time. Three ex-vivo porcine tongues and two cheeks were used with four markers implanted submucosally for each sample. NIR pictures were taken over 26 days. The signal intensities (Fig. 2) and areas of each marker were measured on collected images. SNR was greater than 3.6 over 26 days allowing clear visualization of this marker over the time period. The decrease in both intensity and area were not found to be statistically significant based on ANOVA tests.
The second study tested the feasibility of the markers for in-vivo performance. Three white mice were used with two markers on the right flank for each sample. NIR pictures were taken right after the injections and then the mice were euthanized. The markers (n=6) were clearly visible with an average of 3.8 for SNR. After euthanasia, we cauterized a small area near the markers for each mouse, then acquired NIR pictures again. The new SNR results remained at a high level equaling 3.7. The color and NIR images of a mouse with two markers are shown (Fig. 3).
Conclusions: In conclusion, our NIRF markers are clearly visible over 26 days on cadaver porcine tongues and cheeks. Considering the good performance on mice even after cauterization and the 19-Hz camera framerate, this marking strategy has the potential for real-time intra-operative tracking of tumors for long-term treatment protocols such as chemotherapy and radiation or neoadjuvant chemotherapy. An in-vivo chronic injection study will be performed in the future.
Photo of the Camera system.
Long-term study results for tongues and cheeks.
Color and NIR images of a mouse with two India Ink (top) and two NIRF markers (bottom).