Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Stain-free histopathology by programmable supercontinuum pulses

Nature Photonics volume 10, pages 534–540 (2016)Cite this article

Subjects

Abstract

The preparation, staining, visualization and interpretation of histological images of tissue is well accepted as the gold standard process for the diagnosis of disease. These methods have a long history of development, and are used ubiquitously in pathology, despite being highly time- and labour-intensive. Here, we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic-crystal fibre source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate the collection of optical signatures of the tumour microenvironment, including evidence of mesoscopic biological organization, tumour cell migration and (lymph-) angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mesoscopic organization of biological microstructures revealed in co-localized multiphoton images of two rat mammary specimens and absent in corresponding FFPE–H&E histology images.
Figure 2: Optical signatures in co-localized multiphoton images of five mammary specimens that are absent in corresponding FFPE–H&E histology images.
Figure 3: Optical signatures of local tumour invasion in large-area tri-modal multiphoton images of two rat mammary specimens that are absent in corresponding FFPE–H&E histology images.

References

  1. Titford, M. & Bowman, B. What may the future hold for histotechnologists? Lab. Med. 43, e5–e10 (2012).

    Article  Google Scholar 

  2. Buesa, R. J. Histology: a unique area of the medical laboratory. Ann. Diagn. Pathol. 11, 137–141 (2007).

    Article  Google Scholar 

  3. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  ADS  Google Scholar 

  4. Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).

    Article  ADS  Google Scholar 

  5. Matthews, T. E., Piletic, I. R., Selim, M. A., Simpson, M. J. & Warren, W. S. Pump–probe imaging differentiates melanoma from melanocytic nevi. Sci. Transl. Med. 3, 71ra15 (2011).

    Article  Google Scholar 

  6. Ji, M. et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci. Transl. Med. 5, 201ra119 (2013).

    Article  Google Scholar 

  7. Tao, Y. K. et al. Assessment of breast pathologies using nonlinear microscopy. Proc. Natl Acad. Sci. USA 111, 15304–15309 (2014).

    Article  ADS  Google Scholar 

  8. Lu, F.-K. et al. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 112, 11624–11629 (2015).

    Article  ADS  Google Scholar 

  9. Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nature Biotechnol. 21, 1369–1377 (2003).

    Article  Google Scholar 

  10. Sukumar, S., Notario, V., Martin-Zanca, D. & Barbacid, M. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature 306, 658–661 (1983).

    Article  ADS  Google Scholar 

  11. Chowdary, P. D. et al. Molecular histopathology by spectrally reconstructed nonlinear interferometric vibrational imaging. Cancer Res. 70, 9562–9569 (2010).

    Article  Google Scholar 

  12. Chance, B., Schoener, B., Oshino, R., Itshak, F. & Nakase, Y. Oxidation–reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J. Biol. Chem. 254, 4764–4771 (1979).

    Article  Google Scholar 

  13. Sutton, D. A Textbook of Radiology and Imaging (Churchill Livingstone, 1987).

    Google Scholar 

  14. Weiner, A. M. Femtosecond pulse shaping using spatial light modulators. Rev. Sci. Instrum. 71, 1929–1960 (2000).

    Article  ADS  Google Scholar 

  15. Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).

    Article  Google Scholar 

  16. Provenzano, P. P. et al. Collagen reorganization at the tumor–stromal interface facilitates local invasion. BMC Med. 4, 38 (2006).

    Article  Google Scholar 

  17. Provenzano, P. P. et al. Collagen density promotes mammary tumor initiation and progression. BMC Med. 6, 11 (2008).

    Article  Google Scholar 

  18. Skala, M. C. et al. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc. Natl Acad. Sci. USA 104, 19494–19499 (2007).

    Article  ADS  Google Scholar 

  19. Menendez, J. A. & Lupu, R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nature Rev. Cancer 7, 763–777 (2007).

    Article  Google Scholar 

  20. Le, T. T., Huff, T. B. & Cheng, J. X. Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis. BMC Cancer 9, 42 (2009).

    Article  Google Scholar 

  21. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    Article  ADS  Google Scholar 

  22. Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nature Rev. Mol. Cell Biol. 10, 445–457 (2009).

    Article  Google Scholar 

  23. Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).

    Article  Google Scholar 

  24. Weis, S. M. & Cheresh, D. A. Tumor angiogenesis: molecular pathways and therapeutic targets. Nature Med. 17, 1359–1370 (2011).

    Article  Google Scholar 

  25. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    Article  Google Scholar 

  26. Folkman, J. & Haudenschild, C. Angiogenesis in vitro. Nature 288, 551–556 (1980).

    Article  ADS  Google Scholar 

  27. Stacker, S. A., Achen, M. G., Jussila, L., Baldwin, M. E. & Alitalo, K. Lymphangiogenesis and cancer metastasis. Nature Rev. Cancer 2, 573–583 (2002).

    Article  Google Scholar 

  28. Buesa, R. J. Histology without formalin? Ann. Diagn. Pathol. 12, 387–396 (2008).

    Article  Google Scholar 

  29. Buesa, R. J. Histology without xylene. Ann. Diagn. Pathol. 13, 246–256 (2008).

    Article  Google Scholar 

  30. Fu, D. et al. High-resolution in vivo imaging of blood vessels without labeling. Opt. Lett. 32, 2641–2643 (2007).

    Article  ADS  Google Scholar 

  31. Min, W. et al. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461, 1105–1109 (2009).

    Article  ADS  Google Scholar 

  32. Mahou, P. et al. Multicolor two-photon tissue imaging by wavelength mixing. Nature Methods 9, 815–818 (2012).

    Article  Google Scholar 

  33. González, S. & Tannous, Z. Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma. J. Am. Acad. Dermatol. 47, 869–874 (2002).

    Article  Google Scholar 

  34. Huang, D. et al. Optical coherence tomography. Science 254, 1178–1181 (1991).

    Article  ADS  Google Scholar 

  35. Zhang, H. F., Maslov, K., Stoica, G. & Wang, L. V. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nature Biotechnol. 24, 848–851 (2006).

    Article  Google Scholar 

  36. Liu, Y. et al. Suppressing short-term polarization noise and related spectral decoherence in all-normal dispersion fiber supercontinuum generation. J. Lightw. Technol. 33, 1814–1820 (2015).

    Article  ADS  Google Scholar 

  37. Lozovoy, V. V., Pastirk, I. & Dantus, M. Multiphoton intrapulse interference. IV. Ultrashort laser pulse spectral phase characterization and compensation. Opt. Lett. 29, 775–777 (2004).

    Article  ADS  Google Scholar 

  38. Liu, Y., Tu, H., Benalcazar, W. A., Chaney, E. J. & Boppart, S. A. Multimodal nonlinear imaging by pulse shaping of a fiber supercontinuum from 900 to 1160 nm. IEEE J. Sel. Top. Quantum Electron. 18, 1209–1214 (2012).

    Article  ADS  Google Scholar 

  39. Pegoraro, A. F. et al. Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator. Opt. Express 17, 2984–2996 (2009).

    Article  ADS  Google Scholar 

  40. Arafah, B. M., Finegan, H. M., Roe, J., Manni, A. & Pearson, O. H. Hormone dependency in N-nitrosomethylurea-induced rat mammary tumors. Endocrinology 111, 584–588 (1982).

    Article  Google Scholar 

  41. Crist, K. A., Chaudhuri, B., Shivaram, S. & Chaudhuri, P. K. Ductal carcinoma in situ in rat mammary gland. J. Surg. Res. 52, 205–208 (1992).

    Article  Google Scholar 

  42. Singh, M., McGinley, J. N. & Thompson, H. J. A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human. Lab. Invest. 80, 221–231 (2000).

    Article  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the National Institutes of Health (R01 CA166309 and R01 EB013723), the Danish Council for Independent Research – Technology and Production Sciences (FTP project ALFIE), the European Commission (EU Career Integration Grant 334324 LIGHTER) and by the Max Planck Society.

Author information

Author notes

  1. Haohua Tu and Yuan Liu: These authors contributed equally to this work

Authors and Affiliations

  1. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Haohua Tu, Yuan Liu, Marina Marjanovic, Eric J. Chaney, Youbo Zhao, Sixian You & Stephen A. Boppart

  2. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Yuan Liu, Marina Marjanovic, Sixian You & Stephen A. Boppart

  3. Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz, 55128, Germany

    Dmitry Turchinovich

  4. NKT Photonics A/S, Blokken 84, Birkerød, 3460, Denmark

    Jens K. Lyngsø

  5. DTU Fotonik, Technical University of Denmark, Ørsteds Plads 343, Lyngby, 2800, Denmark

    Jesper Lægsgaard

  6. Department of Materials Science and Engineering, Frederick Seitz Materials Research Laboratory, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    William L. Wilson

  7. Biophotonic Solutions Inc., East Lansing, 48823, Michigan, USA

    Bingwei Xu

  8. Department of Chemistry and Department of Physics, Michigan State University, East Lansing, 48824, Michigan, USA

    Marcos Dantus

  9. Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Stephen A. Boppart

  10. College of Medicine, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Stephen A. Boppart

Authors
  1. Haohua Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dmitry Turchinovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marina Marjanovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jens K. Lyngsø
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jesper Lægsgaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eric J. Chaney
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Youbo Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sixian You
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. William L. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Bingwei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Marcos Dantus
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Stephen A. Boppart
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.T., Y.L. and S.A.B. conceived the idea, performed the analysis and wrote the manuscript. H.T., Y.L. and Y.Z. built the microscope and conducted optical experiments. H.T., D.T. and J.L. tested and improved the long-term stability of the fibre supercontinuum source. J.K.L. fabricated a variety of photonic-crystal fibres for the supercontinuum source. W.L.W. characterized the supercontinuum source. E.J.C., S.Y. and M.M. performed biological experiments. B.X. and M.D. built the pulse shaper. H.T. and S.A.B. obtained funding for this research.

Corresponding authors

Correspondence to Haohua Tu or Stephen A. Boppart.

Ethics declarations

Competing interests

M.D. is the founder of Biophotonic Solutions Inc., and has financial interest in a proprietary pulse shaping technology using multiphoton intrapulse interference phase scan (MIIPS).

Supplementary information

Supplementary information

Supplementary information (PDF 15106 kb)

Supplementary information

Supplementary Movie 1 (MOV 757 kb)

Supplementary information

Supplementary Movie 2 (MOV 381 kb)

Supplementary information

Supplementary Movie 3 (MOV 203 kb)

Supplementary information

Supplementary Movie 4 (MP4 1450 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tu, H., Liu, Y., Turchinovich, D. et al. Stain-free histopathology by programmable supercontinuum pulses. Nature Photon 10, 534–540 (2016). https://doi.org/10.1038/nphoton.2016.94

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2016.94

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing