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Stain-free histopathology by programmable supercontinuum pulses


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.

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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.


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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




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.

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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).

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Tu, H., Liu, Y., Turchinovich, D. et al. Stain-free histopathology by programmable supercontinuum pulses. Nature Photon 10, 534–540 (2016).

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