Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaax5208/DC1

Fig. S1. Clinical trial SLN mapping case in a patient with head and neck melanoma using cRGDY-Cy5.5-PEG-C dots (NCT02106598).

Fig. S2. Development of 700- and 800-nm emitting NIR fluorescent C dots.

Fig. S3. Morphology and purity characterization of MSH-PEG-Cy5.5-C dots and cRGDY-PEG-CW800-C dots.

Fig. S4. The relationship between probe concentration and signal intensity of different probes.

Fig. S5. The bleed-through of MSH-PEG-Cy5.5-C dot probes to the 800-nm channel under the excitation of a 700-nm laser (particle concentration range, 0 to 125 nM).

Fig. S6. The bleed-through of cRGDY-PEG-CW800-C dot probes to the 700-nm channel under the excitation of a 800-nm laser (concentration range, 0 to 250 nM).

Fig. S7. The bleed-through of cocktail C dots (MSH-PEG-Cy5.5-C dot probes and cRGDY-PEG-CW800-C dot) probes to the 700- or 800-nm channels under the combined excitation of 700- and 800-nm lasers (concentration range, 0 to 250 nM).

Fig. S8. Summary of MIP PET images of all seven spontaneous melanoma miniswines (the related MIP short videos are also provided as movies S1 to S7).

Fig. S9. The marking of the expected SLN locations.

Fig. S10. A representative example of multiplexing optical data analysis using the software (Architector Image Viewer, version 1.9.0) from Quest Spectrum.

Fig. S11. Whole-body PET/CT imaging of pig #1.

Fig. S12. Whole-body PET/CT imaging and histological analysis of pig #2.

Fig. S13. Whole-body PET/CT imaging and histological analysis of pig #3.

Fig. S14. Whole-body PET/CT imaging and histological analysis of pig #4.

Fig. S15. Whole-body PET/CT imaging and histological analysis of pig #6.

Fig. S16. Whole-body PET/CT imaging and histological analysis of pig #7.

Fig. S17. IVIS imaging to identify nanoparticles at the tumor site.

Fig. S18. Average body weight of all male mice from the microdose toxicology study.

Fig. S19. Average body weight of all female mice from the microdose toxicology study.

Table S1. Optimization of 800-nm emitted NIR fluorescent C dots.

Table S2. In vivo signal intensity and bleed-through percentage of cocktail C dots excited with a 700-nm laser.

Table S3. Summary of the injection information of all seven spontaneous melanoma miniswines.

Table S4. Summary of PET SUV numbers of all nodes from seven spontaneous melanoma miniswines (~0.5-mm tumor burden was highlighted in green; NA means tumor burden diameter was not available).

Table S5. Microdose toxicology study animal group.

Table S6. Mortality and morbidity summary table of the microdose toxicology study.

Table S7. Hematology parameters in the microdose toxicology study.

Table S8. Clinical chemistry parameters in the microdose toxicology study.

Table S9. Tissues examined microscopically in the microdose toxicology study.

Table S10. Hematology parameters, group 5, male, tumor, vehicle control (saline), day 2.

Table S11. Hematology parameters, group 6, female, tumor, vehicle control (saline), day 2.

Table S12. Hematology parameters, group 7, male, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S13. Hematology parameters, group 8, female, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S14. Hematology parameters, group 9, male, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S15. Hematology parameters, group 10, female, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S16. Hematology parameters, group 11, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S17. Hematology parameters, group 12, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S18. Hematology parameters, group 13, male, no tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S19. Hematology parameters, group 14, female, no tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S20. Hematology parameters, group 15, male, tumor, vehicle control (saline), day 14.

Table S21. Hematology parameters, group 16, female, tumor, vehicle control (saline), day 14.

Table S22. Hematology parameters, group 17, male, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S23. Hematology parameters, group 18, female, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S24. Hematology parameters, group 19, male, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 14.

Table S25. Hematology parameters, group 20, female, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 14.

Table S26. Hematology parameters, group 21, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S27. Hematology parameters, group 22, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S28. Hematology parameters, group 23, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S29. Hematology parameters, group 24, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S30. Hematology parameters, group 5, male, tumor, vehicle control (saline), day 2.

Table S31. Hematology parameters, group 6, female, tumor, vehicle control (saline), day 2.

Table S32. Hematology parameters, group 7, male, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S33. Hematology parameters, group 8, female, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S34. Hematology parameters, group 9, male, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S35. Hematology parameters, group 10, female, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S36. Hematology parameters, group 11, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S37. Hematology parameters, group 12, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 2.

Table S38. Hematology parameters, group 13, male, no tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S39. Hematology parameters, group 14, female, no tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 2.

Table S40. Hematology parameters, group 15, male, tumor, vehicle control (saline), day 14.

Table S41. Hematology parameters, group 16, female, tumor, vehicle control (saline), day 14.

Table S42. Hematology parameters, group 17, male, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S43. Hematology parameters, group 18, female, tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S44. Hematology parameters, group 19, male, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 14.

Table S45. Hematology parameters, group 20, female, tumor, test article cRGDY-PEG-cw800-C dots at 1.2 nmol, day 14.

Table S46. Hematology parameters, group 21, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S47. Hematology parameters, group 22, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S48. Hematology parameters, group 23, male, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Table S49. Hematology parameters, group 24, female, no tumor, test article MSH-PEG-Cy5.5-C dots at 1.2 nmol, day 14.

Movie S1. MIP video of pig #1.

Movie S2. MIP video of pig #2.

Movie S3. MIP video of pig #3.

Movie S4. MIP video of pig #4.

Movie S5. MIP video of pig #5.

Movie S6. MIP video of pig #6.

Movie S7. MIP video of pig #7.

Movie S8. Image-guided multiplexing, pig #5.

Movie S9. Image-guided multiplexing, pig #3.

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