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

Nature Biomedical Engineering volume 1, Article number: 0084 (2017) Cite this article

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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.

Pathologic features provide a gold-standard by which diseases are diagnosed, patient prognoses are determined, and treatment decisions are made1. In a pathology lab, the examination of a tissue biopsy or a surgically excised specimen is most reliably performed by the microscopic examination of tissues that have been formalin-fixed and paraffin-embedded (FFPE), and then thinly sectioned, stained and mounted on glass slides (Supplementary Fig. 1a). This is a labour- and time-intensive process, in which only a small fraction of a specimen is sampled for microscopy. Frozen sectioning enables tissues to be rapidly sectioned, stained and mounted on slides. However, this technique can introduce severe artefacts , especially for tissues that do not freeze well (such as breast tissue). Furthermore, similarly to FFPE histology, it is destructive of tissue and suffers from the same sampling limitations2.

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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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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.

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Author notes

  1. Adam K. Glaser and Nicholas P. Reder: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Mechanical Engineering, University of Washington, Seattle, Washington, 98105, USA

    Adam K. Glaser, Ye Chen, Chengbo Yin, Linpeng Wei, Yu Wang & Jonathan T. C. Liu

  2. Department of Pathology, University of Washington, Seattle, Washington, 98105, USA

    Nicholas P. Reder, Erin F. McCarty & Lawrence D. True

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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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