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Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens

Abstract

For the 1.7 million patients per year in the US who receive a new cancer diagnosis, treatment decisions are largely based on histopathological specimen examinations. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 μm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 s cm−2), and volumetric assessment of optically cleared core-needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens.

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Figure 1: Open-top light-sheet microscope for clinical pathology.
Figure 2: Wide-area surface microscopy of human prostate tissue.
Figure 3: Rapid intraoperative microscopy of human breast tissue.
Figure 4: Volumetric dual-channel imaging of a human biopsy specimen.

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References

  1. Surveillance Research Program, N.C.I. Fast Stats: An Interactive Tool for Access to SEER Cancer Statistics (2016); https://seer.cancer.gov/faststats/

  2. Barakat, F. H., Sulaiman, I. & Sughayer, M. A. Reliability of frozen section in breast sentinel lymph node examination. Breast Cancer 21, 576– 582 (2014).

    Article  Google Scholar 

  3. McKenney, J. K. et al. The potential impact of reproducibility of Gleason grading in men with early stage prostate cancer managed by active surveillance: a multi-institutional study. J. Urol. 186, 465–469 (2011).

    Article  Google Scholar 

  4. Shah, R. B. et al. Diagnosis of Gleason pattern 5 prostate adenocarcinoma on core needle biopsy: an interobserver reproducibility study among urologic pathologists. Am. J. Surg. Pathol. 39, 1242–1249 (2015).

    Article  Google Scholar 

  5. Meyer, J. S. et al. Breast carcinoma malignancy grading by Bloom-Richardson system vs proliferation index: reproducibility of grade and advantages of proliferation index. Mod. Pathol. 18, 1067–1078 (2005).

    Article  Google Scholar 

  6. Tozbikian, G . et al. Atypical ductal hyperplasia bordering on ductal carcinoma in situ: interobserver variability and outcomes in 105 cases. Int. J. Surg. Pathol. 25, 100–107 (2016).

    Article  Google Scholar 

  7. Bedossa, P., Dargere, D. & Paradis, V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology 38, 1449–1457 (2003).

    Article  Google Scholar 

  8. Roberts, N. et al. Toward routine use of 3D histopathology as a research tool. Am. J. Pathol. 180, 1835–1842 (2012).

    Article  Google Scholar 

  9. Carlson, R. O., Amirahmadi, F. & Hernandez, J. S. A primer on the cost of quality for improvement of laboratory and pathology specimen processes. Am. J. Clin. Pathol. 138, 347–354 (2012).

    Article  Google Scholar 

  10. Gareau, D. S. et al. Confocal mosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology. J. Biomed. Opt. 13, 054001 (2008).

    Article  Google Scholar 

  11. Van Royen, M. E. et al. Three-dimensional microscopic analysis of clinical prostate specimens. Histopathology 69, 985–992 (2016).

    Article  Google Scholar 

  12. Fereidouni, F . et al. Microscopy with UV Surface Excitation (MUSE) for slide-free histology and pathology imaging. Proc. SPIE 9318, 93180F (2015).

    Article  Google Scholar 

  13. Wang, M. et al. High-resolution rapid diagnostic imaging of whole prostate biopsies using video-rate fluorescence structured illumination microscopy. Cancer Res. 75, 4032–4041 (2015).

    Article  Google Scholar 

  14. Wang, M. et al. Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy. Sci. Rep. 6, 27419 (2016 ).

    Article  Google Scholar 

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

    Article  Google Scholar 

  16. 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 

  17. Tu, H. et al. Stain-free histopathology by programmable supercontinuum pulses. Nat. Photon. 10, 534–540 (2016).

    Article  Google Scholar 

  18. Olson, E., Levene, M. J. & Torres, R. Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies. Biomed. Opt. Express 7, 3089–3096 (2016).

    Article  Google Scholar 

  19. Jonkman, J. & Brown, C. M. Any way you slice it—a comparison of confocal microscopy techniques. J. Biomol. Tech. 26, 54–65 (2015).

    Google Scholar 

  20. Mertz, J. Optical sectioning microscopy with planar or structured illumination. Nat. Methods 8, 811–819 (2011).

    Article  Google Scholar 

  21. Vakoc, B. J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nat. Med. 15, 1219–1223 (2009).

    Article  Google Scholar 

  22. Assayag, O. et al. Large field, high resolution full-field optical coherence tomography: a pre-clinical study of human breast tissue and cancer assessment. Technol. Cancer Res. Treat. 13, 455–468 (2014).

    Google Scholar 

  23. Zysk, A. M. et al. Optical coherence tomography: a review of clinical development from bench to bedside. J. Biomed. Opt. 12, 051403 (2007).

    Article  Google Scholar 

  24. Siedentopf, H. & Zsigmondy, R. Uber Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Annal. Phys. 315, 1–39 (1902).

    Article  Google Scholar 

  25. Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

    Article  Google Scholar 

  26. Keller, P. J. et al. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

    Article  Google Scholar 

  27. Cella Zanacchi, F. et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat. Methods 8, 1047–1049 (2011).

    Article  Google Scholar 

  28. Huisken, J. et al. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  Google Scholar 

  29. Glaser, A. K., Wang, Y. & Liu, J. T. Assessing the imaging performance of light sheet microscopies in highly scattering tissues. Biomed. Opt. Express 7, 454–466 (2016).

    Article  Google Scholar 

  30. Pitrone, P. G. et al. OpenSPIM: an open-access light-sheet microscopy platform. Nat. Methods 10, 598–599 (2013).

    Article  Google Scholar 

  31. Reynaud, E. G. et al. Guide to light-sheet microscopy for adventurous biologists. Nat. Methods 12, 30–34 (2015).

    Article  Google Scholar 

  32. Kumar, A. et al. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat. Protoc. 9, 2555–2573 (2014).

    Article  Google Scholar 

  33. Strnad, P. et al. Inverted light-sheet microscope for imaging mouse pre-implantation development. Nat. Methods 13, 139–142 (2016).

    Article  Google Scholar 

  34. Yang, Z. et al. An inverted light sheet microscope optimized for studies in neuroscience. Conf. CLEO Atu3O.5 (2016).

  35. Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 108, 17708–17713 (2011).

    Article  Google Scholar 

  36. Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    Article  Google Scholar 

  37. McGorty, R. et al. Open-top selective plane illumination microscope for conventionally mounted specimens. Opt. Express 23, 16142–16153 (2015).

    Article  Google Scholar 

  38. Kino, G. S. Applications and theory of the solid immersion lens. Proc. SPIE 3609, 56 (1999).

    Google Scholar 

  39. 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 

  40. Hall, G. S., Kramer, C. E. & Epstein, J. I. Evaluation of radical prostatectomy specimens. A comparative analysis of sampling methods. Am. J. Surg. Pathol. 16, 315–324 (1992).

    Article  Google Scholar 

  41. Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    Article  Google Scholar 

  42. Elfer, K. N. et al. DRAQ5 and eosin ('D&E') as an analog to hematoxylin and eosin for rapid fluorescence histology of fresh tissues. PLoS ONE 11, e0165530 (2016).

    Article  Google Scholar 

  43. Moffitt, J. R. et al. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc. Natl Acad. Sci. USA 113, 14456–14461 (2016).

    Article  Google Scholar 

  44. Chen, F. et al. Nanoscale imaging of RNA with expansion microscopy. Nat. Methods 13, 679–684 (2016).

    Article  Google Scholar 

  45. Schmid, H. P. & McNeal, J. E. An abbreviated standard procedure for accurate tumor volume estimation in prostate cancer. Am. J. Surg. Pathol. 16, 184–191 (1992).

    Article  Google Scholar 

  46. Sehdev, A. E., Pan, C. C. & Epstein, J. I. Comparative analysis of sampling methods for grossing radical prostatectomy specimens performed for nonpalpable (stage T1c) prostatic adenocarcinoma. Hum. Pathol. 32, 494–499 (2001).

    Article  Google Scholar 

  47. Jacobs, L. Positive margins: the challenge continues for breast surgeons. Ann. Surg. Oncol. 15, 1271–1272 (2008).

    Article  Google Scholar 

  48. Moran, M. S. et al. Society of Surgical Oncology-American Society for Radiation Oncology consensus guideline on margins for breast-conserving surgery with whole-breast irradiation in stages I and II invasive breast cancer. J. Clin. Oncol. 32, 1507–1515 (2014).

    Article  Google Scholar 

  49. Adams, B. J. et al. The role of margin status and reexcision in local recurrence following breast conservation surgery. Ann. Surg. Oncol. 20, 2250–2255 (2013).

    Article  Google Scholar 

  50. Singletary, S. E. et al. Revision of the American Joint Committee on Cancer staging system for breast cancer. J. Clin. Oncol. 20, 3628–3636 (2002).

    Article  Google Scholar 

  51. Zhou, M. et al. Diagnosis of "poorly formed glands" Gleason pattern 4 prostatic adenocarcinoma on needle biopsy: an interobserver reproducibility study among urologic pathologists with recommendations. Am. J. Surg. Pathol. 39, 1331–1339 (2015).

    Article  Google Scholar 

  52. Vettenburg, T. et al. Light-sheet microscopy using an Airy beam. Nat. Methods 11, 541–544 (2014).

    Article  Google Scholar 

  53. Fahrbach, F. O. & Rohrbach, A. Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media. Nat. Commun. 3, 632 (2012).

    Article  Google Scholar 

  54. Fu, Q. et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nat. Commun. 7, 11088 (2016).

    Article  Google Scholar 

  55. De Medeiros, G. et al. Confocal multiview light-sheet microscopy. Nat. Commun. 6, 8881 (2015).

    Article  Google Scholar 

  56. Tomer, R. et al. Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat. Methods 9, 755–763 (2012).

    Article  Google Scholar 

  57. Dean, K. M. et al. Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy. Optica 4, 263–271 (2017).

    Article  Google Scholar 

  58. Munch, B. et al. Stripe and ring artifact removal with combined wavelet—Fourier filtering. Opt. Express 17, 8567–8591 (2009).

    Article  Google Scholar 

  59. Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463–1465 (2009).

    Article  Google Scholar 

  60. Aguet, F., Van De Ville, D. & Unser, M. Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy. IEEE Trans. Image Process. 17, 1144–1153 (2008).

    Article  Google Scholar 

  61. Giacomelli, M. G. et al. Virtual hematoxylin and eosin transillumination microscopy using epi-fluorescence imaging. PLoS ONE 11, e0159337 (2016).

    Article  Google Scholar 

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Acknowledgements

The authors thank N. Sanai of the Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center (Phoenix, Arizona) for providing the human glioma sample, and B. Najafian for providing the human kidney tissue sample. Human breast specimens were provided by the NorthWest BioTrust, which is supported in part by the NCI of the National Institutes of Health (P30CA015704). Human prostate specimens were provided by the GU Specimen Biorepository, University of Washington, which is supported by resources of the Department of Defense Prostate Cancer Research Program (W81XWH-14-2-0183), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), a PO1 NIH grant (PO1 CA163227), and the Institute for Prostate Cancer Research of the University of Washington. This work was also supported by resources from the NIH/NCI (R01 CA175391 and F32 CA213615), the NIH/NIDCR (R01 DE023497), the University of Washington Royalty Research Fund, and a University of Washington CoMotion Innovation Award.

Author information

Authors and Affiliations

Authors

Contributions

A.K.G., N.P.R. and J.T.C.L. designed the studies. A.K.G. and J.T.C.L. designed the open-top light-sheet microscope. A.K.G., Y.C., C.Y. and L.W. fabricated the microscope. A.K.G., N.P.R. and Y.C. imaged the tissue samples. E.F.M. prepared the optically cleared tissue samples. N.P.R. and L.D.T. performed the blinded evaluation of prostate samples. N.P.R. and L.D.T. provided pathological diagnosis of all samples. A.K.G., N.P.R., Y.C., E.F.M., C.Y., L.W., Y.W., L.D.T. and J.T.C.L. prepared the manuscript.

Corresponding author

Correspondence to Jonathan T. C. Liu.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary methods, figures, tables, video captions and references (PDF 3357 kb)

Supplementary Video 1

Ease-of-use and scanning trajectory of the open-top light-sheet microscope. The sample is a fresh prostate tissue slice, stained with 1 mM acridine orange for 20 s, and rinsed for 10 s in 1× PBS. (MP4 6702 kb)

Supplementary Video 2

Open-top light-sheet microscopy imaging dataset from a piece of fresh human prostate tissue. High-magnification regions of benign glands and stroma are shown. (MP4 18966 kb)

Supplementary Video 3

Open-top light-sheet microscopy imaging dataset from a piece of fresh human breast tissue. High-magnification regions of benign lobules and invasive ductal carcinoma are shown (MP4 17755 kb)

Supplementary Video 4

Volumetric visualization of a human prostate core-needle biopsy with two representative zoomed-in (high-magnification) depth-varying image stacks (sagittal) revealing 3D glandular morphology. (MP4 7631 kb)

Supplementary Video 5

Zoomed-in region of a human prostate core-needle biopsy, in which a stack of depth-varying ‘sections’ is shown (at ~5-μm increments) to mimic a stack of conventional H&E–stained tissue sections on glass slides. (MP4 8209 kb)

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Glaser, A., Reder, N., Chen, Y. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat Biomed Eng 1, 0084 (2017). https://doi.org/10.1038/s41551-017-0084

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