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.

Microscopy with ultraviolet surface excitation for rapid slide-free histology

Nature Biomedical Engineering volume 1, pages 957–966 (2017)Cite this article

Subjects

Abstract

Histological examination of tissues is central to the diagnosis and management of neoplasms and many other diseases and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides—a process that can require hours or days, contributes to cost and delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that, within minutes, provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin and eosin histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280 nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA sequencing). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings and to provide opportunities for rapid histology in research.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Rastogi, V. et al. Artefacts: a diagnostic dilemma—a review. J. Clin. Diagn. Res.7, 2408–2413 (2013).

    PubMed  PubMed Central  Google Scholar 

  2. Neil, M. A., Juskaitis, R. & Wilson, T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt. Lett.22, 1905–1907 (1997).

    Article  CAS  Google Scholar 

  3. Dobbs, J. et al. Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies. Breast Cancer Res. Treat.149, 303–310 (2015).

    Article  CAS  Google Scholar 

  4. Tao, Y. K. et al. Assessment of breast pathologies using nonlinear microscopy. Proc. Natl Acad. Sci. USA111, 15304–15309 (2014).

    Article  CAS  Google Scholar 

  5. Kang, D. et al. Endoscopic probe optics for spectrally encoded confocal microscopy. Biomed. Opt. Express4, 1925–1936 (2013).

    Article  Google Scholar 

  6. Orringer, D. A. et al. Rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated Raman scattering microscopy. Nat. Biomed. Eng.1, 0027 (2017).

    Article  Google Scholar 

  7. Liu, J. T. et al. Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture. J. Biomed. Opt.13, 034020 (2008).

    Article  Google Scholar 

  8. Bouchard, M. B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms. Nat. Photon.9, 113–119 (2015).

    Article  CAS  Google Scholar 

  9. Gabriele, M. L. et al. Optical coherence tomography: history, current status, and laboratory work. Invest. Ophthalmol. Vis. Sci.52, 2425–2436 (2011).

    Article  Google Scholar 

  10. Lin, B., Urayama, S., Saroufeem, R. M., Matthews, D. L. & Demos, S. G. Real-time microscopic imaging of esophageal epithelial disease with autofluorescence under ultraviolet excitation. Opt. Express17, 12502–12509 (2009).

    Article  CAS  Google Scholar 

  11. Lin, B., Urayama, S., Saroufeem, R. M., Matthews, D. L. & Demos, S. G. Characterizing the origin of autofluorescence in human esophageal epithelium under ultraviolet excitation. Opt. Express18, 21074–21082 (2010).

    Article  CAS  Google Scholar 

  12. Kather, J. N. et al. New colours for histology: optimized bivariate colour maps increase perceptual contrast in histological images. PLoS ONE10, e0145572 (2015).

    Article  Google Scholar 

  13. Fereidouni, F., Datta-Mitra, A., Demos, S. & Levenson, R. Microscopy with UV surface excitation (MUSE) for slide-free histology and pathology imaging. In Progress in Biomedical Optics and Imaging - Proc. SPIE9318, 93180F (SPIE, 2015).

  14. Ho, D., Fereidouni, F., Levenson, R. M. & Jagdeo, J. Real-time, high-resolution, in vivo characterization of superficial skin with microscopy using ultraviolet surface excitation (MUSE). J. Drugs Dermatol.15, 1344–1346 (2016).

    PubMed  PubMed Central  Google Scholar 

  15. Zeskind, B. J. et al. Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy. Nat. Methods4, 567–569 (2007).

    Article  CAS  Google Scholar 

  16. Zaak, D. et al. Ultraviolet-excited (308 nm) autofluorescence for bladder cancer detection. Urology60, 1029–1033 (2002).

    Article  Google Scholar 

  17. Sharma, A. & Schulman, S. G. Introduction to Fluorescence Spectroscopy (John Wiley & Sons, New York, 1999).

  18. Kenny, K. B. System and methods for generating a brightfield image using fluorescent images. US patent US8639013 B2 (2011).

  19. Garsha, K. et al. Polyfocal interferometric image acquisition. Google patents CA2849985 C (2016).

  20. Hui, P. & Buza, N. Atlas of Intraoperative Frozen Section Diagnosis in Gynecologic Pathology (Springer, Switzerland, 2015).

  21. Foschini, M. P., Baldovini, C., Ishikawa, Y. & Eusebi, V. The value of large sections in surgical pathology. Int. J. Breast Cancer2012, 785947 (2012).

    Article  Google Scholar 

  22. Abels, E. & Pantanowitz, L. Current state of the regulatory trajectory for whole slide imaging devices in the USA. J. Pathol. Inform.8, 23–23 (2017).

    Article  Google Scholar 

  23. Adeyi, O. A. Pathology services in developing countries—the West African experience. Arch. Pathol. Lab. Med.135, 183–186 (2011).

    PubMed  Google Scholar 

  24. Benediktsson, H., Whitelaw, J. & Roy, I. Pathology services in developing countries: a challenge. Arch. Pathol. Lab. Med.131, 1636–1639 (2007).

    PubMed  Google Scholar 

  25. Srinivasan, M., Sedmak, D. & Jewell, S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol.161, 1961–1971 (2002).

    Article  CAS  Google Scholar 

  26. Kapp, J. R. et al. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial. J. Clin. Pathol.68, 111–118 (2015).

    Article  CAS  Google Scholar 

  27. Penland, S. K. et al. RNA expression analysis of formalin-fixed paraffin-embedded tumors. Lab. Invest.87, 383–391 (2007).

    Article  CAS  Google Scholar 

  28. Maes, E. et al. Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives. Amino Acids45, 205–218 (2013).

    Article  CAS  Google Scholar 

  29. Gnanapragasam, V. J. Unlocking the molecular archive: the emerging use of formalin-fixed paraffin-embedded tissue for biomarker research in urological cancer. BJU Int.105, 274–278 (2010).

    Article  Google Scholar 

  30. Ragan, T. et al. Serial two-photon tomography for automated ex vivo mouse brain imaging. Nat. Methods9, 255–258 (2012).

    Article  CAS  Google Scholar 

  31. Hapke, M. R. & Dehner, L. P. The optically clear nucleus. A reliable sign of papillary carcinoma of the thyroid? Am. J. Surg. Pathol.3, 31–38 (1979).

    Article  CAS  Google Scholar 

  32. Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Springer Science + Business Media, Berlin, 2013).

  33. Lechpammer, M. et al. Pathology of inherited manganese transporter deficiency. Annals Neurol.75, 608–612 (2014).

    Article  CAS  Google Scholar 

  34. Woehrer, A. et al. FISH-based detection of 1p 19q codeletion in oligodendroglial tumors: procedures and protocols for neuropathological practice—a publication under the auspices of the Research Committee of the European Confederation of Neuropathological Societies (Euro-CNS). Clin. Neuropathol.30, 47–55 (2010).

    Article  Google Scholar 

  35. Borodina, T., Adjaye, J. & Sultan, M. A strand-specific library preparation protocol for RNA sequencing. Methods Enzymol.500, 79–98 (2011).

    Article  CAS  Google Scholar 

  36. Levin, J. Z. et al. Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods7, 709–715 (2010).

    Article  CAS  Google Scholar 

  37. Bentley, D. R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature456, 53–59 (2008).

    Article  CAS  Google Scholar 

  38. Wang, L., Wang, S. & Li, W. RSeQC: quality control of RNA-Seq experiments. Bioinformatics28, 2184–2185 (2012).

    Article  CAS  Google Scholar 

  39. Wang, L. et al. Measure transcript integrity using RNA-Seq data. BMC Bioinformatics17, 58 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We acknowledge L. Brandi, A. Datta-Mitra, T. McBroom, A. Adelaja, A. Krueger and L. Martinez for helping with the sample preparation and imaging, J. Wilson and D. Peabody for assisting with the tissue procurement, and E. Hillman for providing critical feedback. This work was partially supported by UC Davis Department of Pathology and Laboratory Medicine start-up funds, a UC Davis Science Translation and Innovative Research grant and an unrestricted gift from Agilent Technologies. This work was performed in part under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344.

Author information

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of California Davis Medical Center, Sacramento, CA, USA

    Farzad Fereidouni, Zachary T. Harmany, Miao Tian, Austin Todd, John A. Kintner, Alexander D. Borowsky, John Bishop, Mirna Lechpammer & Richard Levenson

  2. Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, CA, USA

    John D. McPherson

  3. Lawrence Livermore National Laboratory, Livermore, CA, USA

    Stavros G. Demos

  4. University of Rochester, Rochester, NY, USA

    Stavros G. Demos

Authors
  1. Farzad Fereidouni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zachary T. Harmany
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miao Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Austin Todd
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John A. Kintner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John D. McPherson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander D. Borowsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John Bishop
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mirna Lechpammer
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stavros G. Demos
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Richard Levenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.L. and S.G.D. developed the original observations of tissue imaging using fluorescing stains under UV LED excitation. F.F., S.G.D. and R.L. designed and fabricated the MUSE microscope based on the original design by S.G.D. and conducted the wavelength-dependent depth measurements. Z.T.H. was responsible for the colour-mapping software and maintaining the github repository. R.L., F.F., M.T., A.T. and J.A.K. performed the experiments on sample preparation, staining and comparisons with traditional histology. R.L., M.T., J.A.K. and M.L. designed and performed the comparison of MUSE and traditional histology in the central nervous system cases. R.L. and A.T. designed and performed the comparison of MUSE tissues with traditional histology, which were viewed and interpreted by J.B. and A.D.B. R.L., F.F., M.L. and J.D.M. designed and performed the FISH and RNA-Seq experiments. All authors discussed and interpreted the results and wrote and edited the paper.

Corresponding author

Correspondence to Richard Levenson.

Ethics declarations

Competing interests

R.L. and S.G.D. are co-founders of a start-up company, Muse Microscopy, which is involved in commercializing MUSE technology. The other authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary figures, tables, notes and references

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fereidouni, F., Harmany, Z.T., Tian, M. et al. Microscopy with ultraviolet surface excitation for rapid slide-free histology. Nat Biomed Eng 1, 957–966 (2017). https://doi.org/10.1038/s41551-017-0165-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-017-0165-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer