Answer to REVIEWER

that this study tracks the distribution of Q-dots within normal nude mice. Why is it termed the sentinel node if there is no tumor from which drains to it? Consider changing the wording to regional draining lymph nodes instead of sentinel lymph nodes which are more commonly defined as the hypothetical first group of nodes reached by metastasizing cancer cells from a tumor.” Answer: We are sorry for this confusion. We wanted to assess the lymphotropism of QDs before developing a cancer model. Moreover, when SLN is highly metastatic the technique could not be used because of the blockage of lymphatics. Otherwise, the kinetic of accumulation in SLN would be concerned but not the whole distribution of QDs in the body. However, we intended to evaluate the biodistribution of QDs in a metastatic tumor model. Modifications: We specified the use of healthy mice in the title and the abstract of the manuscript and we replaced the term “sentinel lymph node” by “axillary lymph node”. Reviewer comments: “Another concern is that they were unable to get tighter error bars in Figure 4. Especially when this is to be ultimately used clinically. It was also unfortunate that they were unable to detect a difference between PBS and Q-dots without having to make and incision in the mouse. This is NOT a non-Invasive technique as stated in the paper. It seems that this technique has it's advantages in being able to sensitively detect as little as 20 pmol of Q-dots within 5 minutes after injection. However the limitations may outweigh this perk in not being able to detect Q-dots without making several incisions through the skin (not clinically friendly). Answer: We agree with these comments. However, non-invasive measurements are frequently suffering from significant variations of measurements, related to light diffusion and scattering. The results have been obtained from 3-5 mice, being sufficient for statistical analysis. Further, actually, our work is a preliminary step to the use of NIR emitting QDs with which we expect the detection of QDs through the skin without any incision. Till then, we replaced the termination “non invasive” by “minimally invasive”. Modifications: The following sentence was modified in abstract section (p. 4): Conclusions: This work was done using very low doses of injected QDs and the detection was done using a minimally invasive method. The following sentence was modified in Conclusion section (p. 16): A strong fluorescence signal is obtained within few minutes lasting at least till 24 h as measured with optical fiber spectrofluorometry, a minimally invasive technique allowing rapid per-operative complete procedure. Reviewer comments: “From a personal prospective, I feel that the authors need to be more convincing in explaining why this method would be preferred over the existing lymphoscitigraphy method with radioactive 99mTc. Their proposed method with Q-dots has serious depth of penetration limitations unlike radioactive 99mTc, and the radiation dose that one receives for this study is well understood and not deemed "dangerous". However the unknown toxicity effects of the Q-dots remains to be seen, especially if the majority of the Qdots are staying near the injection site as stated in this paper. Overall the reasoning to replace the current clinical methods with this Q-dot technique is not convincing. The authors reasoning is that radioactive material is "unsafe" and that blue dye can cause anaphylactic shock and unflattering blue staining, but the advantages they list for Q-dots (resistance to photobleaching, high quantum yield and cross section) are advantages over other optical probes, not radiation and blue dye. It needs to be worded better. It is still unknown how toxic Q-dots will be in humans, you can't claim that radiation and blue dye are "unsafe" (yet they are used in the clinic) and suggest that q-dots are, even though it is still unknown.” Answer: We agree with the reviewer. Sentinel lymph node biopsy (SLNB) is elegantly simple in concept but quite often very difficult to perform accurately in individual patients. Current techniques of sentinel lymph node (SLN) mapping in breast cancer and melanoma involve preoperative injection of a radioactive colloid tracer (e.g., 99mTc sulfur colloid) followed by intraoperative injection of a visible blue dye (e.g., isosulfan blue). The dye allows limited visualization of afferent lymphatic vessels and the SLN, and the radioactive colloid tracer improves detection rate and confirms complete collection of the SLN with the use of an intraoperative handheld gamma probe. Optical imaging using the fluorescence of quantum dots would appear to have immediate advantages in SLNB over current methods. Surgeons would not require retraining to apply this method as it is similar to the current intraoperative use of blue dye. The advantage over blue dye is that there would be real-time visual guidance of the surgery, with resolution being limited only by the visual acuity of the surgeon. Visual inspection would also allow the surgeon to confirm that all sentinel nodes had been removed from the node field. There would be no need for a gamma probe to confirm this and no need for blue dye injection. There is also the exciting potential for the histopathologist to focus exactly on the part of the sentinel node containing the quantum dots by using a fluorescent microscope. This is precisely the part of the sentinel node that would contain any micrometastasis. Future research could be directed to examining whether the presence of quantum dots in sentinel nodes can be determined retrospectively in a histological specimen. When a SLNB failure occurs (i.e., the tumor recurs in a node field that had a negative SLNB procedure), it is very important to know whether the true sentinel node was removed at the time of surgery, and a measure of the quantity of quantum dots in the node would help this determination. For SLNB in melanoma and breast cancer using quantum dots, we would see preoperative lymphoscintigraphy using a radiolabeled colloid to provide an accurate map of lymph drainage from the tumor site with the exact surface location of the sentinel nodes being marked on the skin. Sometimes lymph drainage is slow and can take up to three hours, an observational period that would be unacceptable in an operating suite (specially in ambulatory surgery). SLNB could then be performed using optical imaging with quantum dots; this would not need to be performed within 24 hours of the lymphoscintigraphy, as is currently the case when an intraoperative gamma-detecting probe is used (owing to the rapid radioactive decay of the isotope). This would simplify scheduling of operating theater time. At present, surgeons attempting SLNB in cancers involving visceral organs such as the gastrointestinal tract, lung, prostate and uterus encounter significant technical difficulties. Performing preoperative lymphoscintigraphy in these patients is extremely difficult, if not impossible, and it is in these patients where we see this exciting new approach having its greatest impact. There would be no need to perform any preoperative mapping, and, with the operative field on view, the surgeon would be able to rapidly detect the sentinel nodes. Bertagnolli et al. (Journal of Clinical Oncology, 2006;24:878-83) found that the SLNB procedureis not as accurate for colon cancer as it is for breast cancer or malignant melanoma. Significant limitations of that study, including low patient number,lack of standardization of injection technique, and technical complexity, highligh the inadequacies of the current SLNB mapping technology. Light-guided sentinel lymph node mapping provides unequivocal, realtime identification of the sentinel lymph node, thus eliminating technical errors that lead to false-negative results. In addition, it will be important to thoroughly assess the potential toxicity of quantum dots. Because QDs are ideally suited for imaging live cells and organisms over long periods, it is imperative that QD labeling is not deleterious to cells. The presence of cadmium and selenium in the core of QDs led to the belief that QDs are toxic; however, QDs have shown no toxicity in live animals when injected into the bloodstream of pigs and for up to 4 months in mice. Even when QDs were loaded in cells growing in vitro, no toxicity was detected after 2 weeks of growth. Furthermore, no deleterious effects of QDs in cells have been observed in vivo in experiments using Xenopus, Dictyostelium and mouse. In these studies, various cellular behaviors including the growth, development, signaling, chemotaxis, differentiation and, in the case of tumor cells, extravasation and nodule formation of Qds labeled cells were indistinguishable from those of unlabeled cells. These results indicate that QDs are inert and safe for live cell labeling and tracking over long periods. Nevertheless, whenever a new approach for QD synthesis or coating is used, or if QDs are used in an extreme environment that could compromise their integrity, it is important to test for their cytotoxicity. Overall, the use of quantum dot imaging promises to contribute significantly to the surgical management of cancer patients. It should have a major impact in SLNB of visceral cancers and also the potential to simplify SLNB in melanoma and breast cancer. Modifications: The following sentence was added in Background section (p. 5-6): Despite the significant improvement of SLN biopsy over ALDN, the problems related to potential radiation hazards along with an extra cost of the radioisotope, to allergic reactions after blue dye injection and the overall procedure duration are limiting factors for this technique [6]. The ideal method should be sensitive, accurate, rapid, non invasive, non radioactive, and potentially usable in a laparoscopic setting for gastrointestinal tumors [7]. None of the current methods fulfils all of these criteria. Lymphatic mapping with blue dye results in a high rate of false-positive nodes because the small dye particles can readily diffuse through the true SLN and traverse multiple nodes [8]. Additionally, blue dye has poor tissue contrast and is difficult to detect in deep, dark anatomical regions such as the abdomen. Although the use of radioisotope tracers has improved the detection rate and accuracy of SLN mapping, the high radioactivity of the primary injection site can interfere with intraoperative in vivo detection of nearby nodes [3]. Additionally, if radioisotopes are injected during surgery, the time period required for the tracer to migrate to the SLN may delay the operative procedure. Conversely, preoperative injection of the radiocolloid tracer often necessitates anywhere from 2 to 16 hours before surgery. This is difficult, if not impossible, to accomplish and is applicable only in endoscopically accessible regions of the colon and stomach [7]. The following sentence was modified in Discussion section (p. 15): Furthermore, to avoid toxicity caused by biodegradation of QDs and exposition of core metal ions, QDs with nonheavy metals cores (such as indium) would be developed as well as ameliorations of surface chemistry [30]. Minor Essential Revisions Reviewer comments: “Aside from the grammar in the text, Table 2 is mislabeled where the numbers have commas (,) instead of decimal points(.). Answer: We are sorry for this overlook and we corrected it in Table 2. Modifications: Please, see the Table 2 Discretionary Revisions Reviewer comments: “What about other lymph nodes in the body, were they taken for q-dot evaluation? It would be interesting to see how far the Q-dots could travel through the lymphatics. Can Q-dots be labeled with blue dye? It's always good to have an extra way of validating Q-dot biodistribution.” Answer: We didn’t evaluate other lymph nodes but it would be highly probable that QDs migrate through following nodes. It was interesting to evaluate this migration since we see a decrease in the amount of QDs in ALN between 60 min and 24 h post-injection. However, it would be done on bigger animals with a wider lymphatic system like rat and pig. Concerning the validation of QDs biodistribution, we intended to label QDs with fluorodeoxyglucose (FDG) or some others markers to track their localisation by microPET. Modifications: no modification Reviewer comments: “Again, the author needs to more concisely organize their thoughts on how this method will be advantageous over the current lymphoscintigraphy method. There are several news articles that describe it better in laymenâ##s terms.” Answer: Please, see the answer and the modifications in the Major Compulsory Revisions in response to the fourth Reviewer comments. Reviewer comments: “The author's don't discuss much about the fluorescence excitation laser fiber optic probe. Is the energy high on this laser where it will destroy surrounding tissue?” Answer: The energy of the fiber optical probe is very low, approximately 0.4 mW/cm 2 , which isn’t hazardous for the surrounding tissue. Modifications: no modification