Abstract
The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.
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Data availability
All data sets are included in this article and its Supplementary Information files. Pathology scoring for Figs. 3d and 4e was conducted on the basis of visual inspection of the slides by expert breast cancer pathologists using an Olympus BX51 optical microscope. Histology images used for this study are available in figshare with the identifier https://doi.org/10.6084/m9.figshare.14897697.v2. Source data are provided with this paper.
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Acknowledgements
B.A., E.B. and K.A.N. acknowledge the support of the Australian Research Council through the Centre of Excellence in Advanced Molecular Imaging (CE140100011). Fellowship funding from the Victorian Cancer Agency and grant funding from the National Breast Cancer Foundation NBCF (IIRS-21-069) is acknowledged by B.S.P. Funding from the National Breast Cancer Foundation (NBCF Prac. 16-006) and Sydney Breast Cancer Foundation is acknowledged by S.O’T. B.A. acknowledges support from the La Trobe Biomedical and Environmental Sensor Technology (BEST) Research Centre. The authors gratefully acknowledge X. Li from the La Trobe Statistics Consultancy Platform for help and advice with the statistical analysis of the data. This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF).
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Contributions
E.B. carried out the research, performed the measurements, analysed the data and generated the figures for the manuscript. E.B., B.S.P. and B.A. conceived the idea and are responsible for the conceptual design of the study. E.B. and B.A. jointly developed the original concept for ‘nanoslide’ and ‘histoplasmonics’. B.S.P. was responsible for design and management of cancer histological studies. B.M. and B.Y. sourced patient samples and assisted with diagnostic sample identification for study. C.S. assisted with experiments. E.H., B.S.P. and A.S. prepared the biological samples and microtome slices. J.O. prepared the optic nerve samples. S.O’T. and B.S.P. performed the pathology scoring and, along with K.H., prepared patient samples. B.A. directed the project. K.A.N. discussed the results and analysis. B.A., B.S.P. and E.B. wrote the paper. All authors discussed the results and contributed to the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Finite element methods (FEM) optical transmission simulations.
a, FEM model of a section of the nanoslide showing the glass substrate (dark blue), nanoaperture in the metallic film, the capping layer, and air (light blue) constructed in the COMSOL Multiphysics software program. Simulated transmission spectra from each of the four lines (I-IV) which make up the chevron sample (see Fig. 2) and the background (B) spectrum generated using b, FEM model of the device for circular apertures (diameter = 160 nm). c, Shows the simulated background spectrum (no sample) for the crosses (arm length = 160 nm), ellipse (major axis = 210 nm, minor axis = 160 nm), and circular apertures. The peaks represent the plasmon resonances that occur within the active layer of the device. d, Example FEM simulations of a cross section of the electric field generated by a single circular nanoaperture at the peak resonant wavelength of the spectra shown in b
Extended Data Fig. 2 AFM line-scan height profiles of the fabricated samples.
a, Schematics of the chevron structure and b, the ‘staircase’ sample. c, The AFM line-scan height profile of the chevron structure sample, the thinnest sample stripe is 3 ± 1 nm. d, The AFM line-scan height profile across the steps and stripes respectively of the ‘staircase’ sample
Extended Data Fig. 3 Pathology workflow for small-animal study and schematic showing serial sections used.
a, Pathology workflow for small-animal, MMTV-PyMT mouse model study. b, Schematic showing how serial sections were taken to enable a direct comparison of nanoslide, H&E, and Ki67.
Extended Data Fig. 4 Large field-of-view example images from H&E, Ki67, and nanoslide.
The Ki67 and nanoslide positive areas are overlaid. Large-field-of-view (3.8 × 3.8 mm2) areas of tissue stained with H&E and Ki67 compared to the results obtained directly on nanoslide. The areas of positivity of both nanoslide (light blue) and Ki67 (bright green) were identified based on their respective HSL colour space values for example, ref. 32 (also see Methods).
Extended Data Fig. 5 Comparison of histoplasmonics to H&E staining of different tissues.
Bright-field optical images: H&E stained images and histoplasmonic images. Tissue slices were placed on the slides in a sequential manner.
Extended Data Fig. 6 Colorimetric analysis and pathology assessment of mouse data.
a, Histology images (200× magnification) were sub-categorized into four different stages for both nanoslide (1st column) and Ki67 (3rd column). The HSL image pixel colour space values were compared against ground truth pathology annotations and classified as True Positive (TP - green), False Negative (FN - red), False Positive (FP - yellow), and True Negative (TN - blue). The white space in the Ki67 image in the top row is an area where no stain adhered. Scale bars = 15 μm. b, H&E images for neoplastic regions –yellow outline (1st column), nanoslide intensity (2nd column), and Ki67 (3rd column) positivity.
Extended Data Fig. 7 Cell counting Ki67 vs nanoslide using brightfield microscopy.
Example histoplasmonic images of 1 μm thick sequential cancerous breast tissues (PyMT mice). a, Low magnification ( × 100) images. Left: contouring of cancerous regions on Nanoslide. Middle: contouring of the same cancerous regions using Ki67. Right: the contours for Nanoslide (blue) is overlapped with the contour for Ki67 (red). b, High magnification ( × 200) images. Left: Nanoslide image of MIN region; Middle: Ki67 image of the same region. Right: Overlap of positive cells on Nanoslide and MIN (92% concordance). c, High magnification (×200) images. Left: Nanoslide image of MIN region; Middle: Ki67 image of the same region. Right: Overlap of positive cells on Nanoslide and MIN (93% concordance).
Extended Data Fig. 8 Schematic showing serial sections used for DCIS breast cancer patient study.
Schematic showing how serial sections were taken to enable a direct comparison of nanoslide, H&E, ER, and CK 5/6 for human tissue.
Extended Data Fig. 9 Bright-field microscopy of 70 nm thick TEM optic nerve tissue sections imaged using toluidine staining and with histoplasmonics.
Top: Bright-field optical image of toluidine stained 70 nm thick optic nerve sections. Nanoslide results: middle – at 0° incident polarization, bottom – at 90° incident polarization reveals axons, glia and the myelin sheath as different colours. The black scale bar in both the left and right column images is 5 μm.
Extended Data Fig. 10 Bright-field microscopy of tissues of different thicknesses.
Left: Histoplasmonic images of cancerous breast tissues (PyMT mice) sectioned at 4 and 5 μm slice thickness showing almost no difference in colour contrast. Right: Bright-field optical images of optic nerve sections sectioned at 70 and 200 nm thickness showing dramatic difference in colour contrast. The scale bar is 15 μm.
Supplementary information
Supplementary Information
This file contains Supplementary Figures 1-6 and Supplementary Tables 1-4.
Supplementary Video 1
Example video of pathologist using nanoslide for histoplasmonics. Video shows an example of breast tissue being examined on nanoslide. Note that the colours as they appear in the video look slightly different to when images are captured directly using the microscope.
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Balaur, E., O’ Toole, S., Spurling, A.J. et al. Colorimetric histology using plasmonically active microscope slides. Nature 598, 65–71 (2021). https://doi.org/10.1038/s41586-021-03835-2
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DOI: https://doi.org/10.1038/s41586-021-03835-2
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