Tackling standardization in fluorescence molecular imaging


The emerging clinical use of targeted fluorescent agents heralds a shift in intraoperative imaging practices that overcome the limitations of human vision. However, in contrast to established radiological methods, no appropriate performance specifications and standards have been established in fluorescence molecular imaging. Moreover, the dependence of fluorescence signals on many experimental parameters and the use of wavelengths ranging from the visible to short-wave infrared (400–1,700 nm) complicate quality control in fluorescence molecular imaging. Here, we discuss the experimental parameters that critically affect fluorescence molecular imaging accuracy, and introduce the concept of high-fidelity fluorescence imaging as a means for ensuring reliable reproduction of fluorescence biodistribution in tissue.

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Fig. 1: Fluorescence intraoperative imaging.
Fig. 2: Fluorescence imaging challenges.
Fig. 3: Effects of tissue optical properties on the fluorescence (and back-reflected) intensity.


  1. 1.

    Koch, M. & Ntziachristos, V. Advancing surgical vision with fluorescence imaging. Annu. Rev. Med. 67, 153–164 (2016).

    Article  Google Scholar 

  2. 2.

    Zhang, R. R. et al. Beyond the margins: real-time detection of cancer using targeted fluorophores. Nat. Rev. Clin. Oncol. 14, 347–364 (2017).

    Article  Google Scholar 

  3. 3.

    van Dam, G. M. et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat. Med. 17, 1315–1319 (2011).

    Article  Google Scholar 

  4. 4.

    Tummers, Q. R. J. G., Hoogstins, C. E. S. & Gaarenstroom, K. N. Intraoperative imaging of folate receptor alpha positive ovarian and breast cancer using the tumor specific agent EC17. Oncotarget 7, 32144–32155 (2016).

    Article  Google Scholar 

  5. 5.

    Rosenthal, E. L. et al. Safety and tumor specificity of cetuximab-IRDye800 for surgical navigation in head and neck cancer. Clin. Cancer Res. 21, 3658–3666 (2015).

    Article  Google Scholar 

  6. 6.

    Whitley, M. J. et al. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer. Sci. Transl. Med. 8, 320ra4 (2016).

    Article  Google Scholar 

  7. 7.

    Hsiung, P.-L. et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat. Med. 14, 454–458 (2008).

    Article  Google Scholar 

  8. 8.

    Atreya, R. et al. In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn’s disease. Nat. Med. 20, 313–318 (2014).

    Article  Google Scholar 

  9. 9.

    Glatz, J., Symvoulidis, P., Garcia-Allende, P. B. & Ntziachristos, V. Robust overlay schemes for the fusion of fluorescence and color channels in biological imaging. J. Biomed. Opt. 19, 040501 (2014).

    ADS  Article  Google Scholar 

  10. 10.

    Glatz, J. et al. Concurrent video-rate color and near-infrared fluorescence laparoscopy. J. Biomed. Opt. 18, 101302 (2013).

    ADS  Article  Google Scholar 

  11. 11.

    Hong, G. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photon. 8, 723–730 (2014).

    ADS  Article  Google Scholar 

  12. 12.

    Hong, G. et al. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 18, 1841–1846 (2012).

    Article  Google Scholar 

  13. 13.

    Ghosh, D. et al. Deep, noninvasive imaging and surgical guidance of submillimeter tumors using targeted M13-stabilized single-walled carbon nanotubes. Proc. Natl Acad. Sci. USA 111, 13948–13953 (2014).

    ADS  Article  Google Scholar 

  14. 14.

    Carr, J. A. et al. Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green. Proc. Natl Acad. Sci. USA 115, 4465–4470 (2018).

    Article  Google Scholar 

  15. 15.

    DSouza, A. V., Lin, H., Henderson, E. R., Samkoe, K. S. & Pogue, B. W. Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging. J. Biomed. Opt. 21, 080901 (2016).

    ADS  Article  Google Scholar 

  16. 16.

    Scheuer, W., van Dam, G. M., Dobosz, M., Schwaiger, M. & Ntziachristos, V. Drug-based optical agents: infiltrating clinics at lower risk. Sci. Transl. Med. 4, 134ps11 (2012).

    Article  Google Scholar 

  17. 17.

    Marshall, M. V. et al. Near-infrared fluorescence imaging in humans with indocyanine green: a review and update. Open Surg. Oncol. J. 2, 12–25 (2012).

    Article  Google Scholar 

  18. 18.

    US National Library of Medicine. VEGF-targeted fluorescent tracer imaging in breast cancer. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT01508572 (2012).

  19. 19.

    Bradley, R. S. & Thorniley, M. S. A review of attenuation correction techniques for tissue fluorescence. J. R. Soc. Interface 3, 1–13 (2006).

    Article  Google Scholar 

  20. 20.

    Themelis, G., Yoo, J. S., Soh, K.-S., Schulz, R. & Ntziachristos, V. Real-time intraoperative fluorescence imaging system using light-absorption correction. J. Biomed. Opt. 14, 064012 (2014).

    Article  Google Scholar 

  21. 21.

    Moriyama, E. H., Kim, A., Bogaards, A., Lilge, L. & Wilson, B. C. A ratiometric fluorescence imaging system for surgical guidance. Adv. Opt. Technol. 2008, 532368 (2008).

    Article  Google Scholar 

  22. 22.

    DeWerd, L. A. & Kissick, M. The Phantoms of Medical and Health Physics (Springer, New York, NY, 2014).

  23. 23.

    Zhu, B., Rasmussen, J. C., Litorja, M. & Sevick-Muraca, E. M. Determining the performance of fluorescence molecular imaging devices using traceable working standards with SI units of radiance. IEEE Trans. Med. Imaging 35, 802–811 (2016).

    Article  Google Scholar 

  24. 24.

    Ntziachristos, V. et al. Planar fluorescence imaging using normalized data. J. Biomed. Opt. 10, 064007 (2005).

    ADS  Article  Google Scholar 

  25. 25.

    Tichauer, K. M. et al. Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging. Nat. Med. 20, 1348–1353 (2014).

    Article  Google Scholar 

  26. 26.

    Valdés, P. A. et al. Quantitative, spectrally-resolved intraoperative fluorescence imaging. Sci. Rep. 2, 798 (2012).

    Article  Google Scholar 

  27. 27.

    Zhu, B., Rasmussen, J. C. & Sevick-Muraca, E. M. Non-invasive fluorescence imaging under ambient light conditions using a modulated ICCD and laser diode. Biomed. Opt. Express 5, 562–572 (2014).

    Article  Google Scholar 

  28. 28.

    Sexton, K. et al. Pulsed-light imaging for fluorescence guided surgery under normal room lighting. Opt. Lett. 38, 3249–3252 (2013).

    ADS  Article  Google Scholar 

  29. 29.

    Zonios, G. & Dimou, A. Modeling diffuse reflectance from semi-infinite turbid media: application to the study of skin optical properties. Opt. Express 14, 8661–8674 (2006).

    ADS  Article  Google Scholar 

  30. 30.

    Garcia-Allende, P. B. et al. Uniqueness in multispectral constant-wave epi-illumination imaging. Opt. Lett. 41, 3098–3101 (2016).

    ADS  Article  Google Scholar 

  31. 31.

    Saager, R. B., Cuccia, D. J., Saggese, S., Kelly, K. M. & Durkin, A. J. Quantitative fluorescence imaging of protoporphyrin IX through determination of tissue optical properties in the spatial frequency domain. J. Biomed. Opt. 16, 126013 (2011).

    ADS  Article  Google Scholar 

  32. 32.

    Yang, B. & Tunnell, J. W. Real-time absorption reduced surface fluorescence imaging. J. Biomed. Opt. 19, 090505 (2014).

    ADS  Article  Google Scholar 

  33. 33.

    Kanick, S. C. & Pogue, B. W. Why reflectance is an imperfect basis for the correction of fluorescence distortion due to optical properties. In Biomedical Optics 2014 BS3A.35 (OSA, 2014).

  34. 34.

    Bogaards, A., Sterenborg, H. J. C. M. & Wilson, B. C. In vivo quantification of fluorescent molecular markers in real-time: a review to evaluate the performance of five existing methods. Photodiagnosis Photodyn. Ther. 4, 170–178 (2007).

    Article  Google Scholar 

  35. 35.

    Yang, V. X. D., Muller, P. J., Herman, P. & Wilson, B. C. A multispectral fluorescence imaging system: design and initial clinical tests in intra-operative photofrin-photodynamic therapy of brain tumors. Lasers Surg. Med. 32, 224–232 (2003).

    Article  Google Scholar 

  36. 36.

    Minamikawa, T. et al. Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions. Sci. Rep. 6, 25530 (2016).

    ADS  Article  Google Scholar 

  37. 37.

    Haller, J. et al. Visualization of pulmonary inflammation using noninvasive fluorescence molecular imaging. J. Appl. Physiol. 104, 795–802 (2008).

    Article  Google Scholar 

  38. 38.

    Godavarty, A. et al. Fluorescence-enhanced optical imaging in large tissue volumes using a gain-modulated ICCD camera. Phys. Med. Biol. 48, 1701–1720 (2003).

    Article  Google Scholar 

  39. 39.

    Vervandier, J. & Gioux, S. Single snapshot imaging of optical properties. Biomed. Opt. Express 4, 2938–2944 (2013).

    Article  Google Scholar 

  40. 40.

    Niedre, M. J. et al. Early photon tomography allows fluorescence detection of lung carcinomas and disease progression in mice in vivo. Proc. Natl Acad. Sci. USA 105, 19126–19131 (2008).

    ADS  Article  Google Scholar 

  41. 41.

    Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R. & Nann, T. Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 5, 763–775 (2008).

    Article  Google Scholar 

  42. 42.

    Gao, X. et al. In vivo molecular and cellular imaging with quantum dots. Curr. Opin. Biotechnol. 16, 63–72 (2005).

    Article  Google Scholar 

  43. 43.

    Alivisatos, A. P., Gu, W. & Larabell, C. Quantum dots as cellular probes. Annu. Rev. Biomed. Eng. 7, 55–76 (2005).

    Article  Google Scholar 

  44. 44.

    Zhu, B., Tan, I.-C., Rasmussen, J. C. & Sevick-Muraca, E. M. Validating the sensitivity and performance of near-infrared fluorescence imaging and tomography devices using a novel solid phantom and measurement approach. Technol. Cancer Res. Treat. 11, 95–104 (2012).

    Article  Google Scholar 

  45. 45.

    Roy, M., Kim, A., Dadani, F. & Wilson, B. C. Homogenized tissue phantoms for quantitative evaluation of subsurface fluorescence contrast. J. Biomed. Opt. 16, 016013 (2011).

    ADS  Article  Google Scholar 

  46. 46.

    Zhu, B., Rasmussen, J. C. & Sevick-Muraca, E. M. A matter of collection and detection for intraoperative and noninvasive near-infrared fluorescence molecular imaging: to see or not to see? Med. Phys. 41, 022105 (2014).

    Article  Google Scholar 

  47. 47.

    Moffitt, T., Chen, Y.-C. & Prahl, S. A. Preparation and characterization of polyurethane optical phantoms. J. Biomed. Opt. 11, 041103 (2006).

    ADS  Article  Google Scholar 

  48. 48.

    Anastasopoulou, M. et al. Comprehensive phantom for interventional fluorescence molecular imaging. J. Biomed. Opt. 21, 091309 (2016).

    ADS  Article  Google Scholar 

  49. 49.

    Gorpas, D., Koch, M., Anastasopoulou, M., Klemm, U. & Ntziachristos, V. Benchmarking of fluorescence cameras through the use of a composite phantom. J. Biomed. Opt. 22, 016009 (2017).

    ADS  Article  Google Scholar 

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We thank A. Ghazaryan for help with optoacoustic measurements. V.N. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 687866 (INNODERM) and from the Deutsche Forschungsgemeinschaft (DFG), Germany (Gottfried Wilhelm Leibnitz Prize 2013; NT 3/10-1).

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Correspondence to Vasilis Ntziachristos.

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After the completion of the manuscript, but before the final submission, Bracco Imaging Deutschland, a company commercializing targeted fluorescence imaging, employed M.K.

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Koch, M., Symvoulidis, P. & Ntziachristos, V. Tackling standardization in fluorescence molecular imaging. Nature Photon 12, 505–515 (2018). https://doi.org/10.1038/s41566-018-0221-5

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