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PRINCIPAL PUBLICATION AND AUTHORS

Multielement Z-tag imaging by X-ray fluorescence microscopy for next-generation multiplex imaging, M. Strotton (a,b), T. Hosogane (a,b,c), M. di Michiel (d), H. Moch (e), Z. Varga (e), B. Bodenmiller (a,b), Nat. Methods 20, 1310-1322 (2023); https:/doi.org/10.1038/s41592-023-01977-x (a) Department of Quantitative Biomedicine, University of Zürich (Switzerland) (b) Institute for Molecular Health Sciences, ETH Zürich (Switzerland) (c) Life Science Zürich Graduate School, ETH Zürich and University of Zürich (Switzerland) (d) ESRF (e) Department of Pathology and Molecular Pathology, University Hospital Zürich (Switzerland)

REFERENCES

[1] L. Kuett et al., Nat. Cancer 3, 122-133 (2022).

scanned using XRF with a 500 nm-diameter X-ray beam at beamline ID15A. As the high-energy X-rays do not interact with the light atoms constituting biological tissue, there is no tissue destruction the X-rays only cause the heavy isotopes to fluoresce. Figure 8 illustrates the principle of the technique.

MEZ-XRF scans gave data comparable to those given by IMC, but were non-destructive and considerably faster about 1000 Hz (pixels per second), versus 200 Hz for IMC. In breast cancer images, it was possible to identify several different cell types at submicron resolution in three different types of tumour. This kind of sample phenotyping is key to determine a patient s course of treatment. For example, if the "HER2" tags are detectable, Herceptin may be prescribed, or immunotherapy might be considered if the immune-cells are present within an inflamed tumour (Figure 9). There is potential for further development by harnessing the unique aspects of X-rays and X-ray fluorescent probes. Improved tag density by

signal amplification combined with improved detectors and further enhanced X-ray flux should advance both detection of the markers and imaging speed. Importantly, since MEZ-XRF builds on the non-destructive penetrating properties of X-rays, it is an ideal approach to image whole tumour samples in 3D.

Such multiscale, multiplexed imaging within a single platform makes new experiments possible. For instance, MEZ-XRF could be used to study signalling and physical interactions between neighbouring rare cell types, which must first be localised in a larger tissue. More generally, the non-destructive multiscale nature of MEZ- XRF means that low-resolution overviews can be used to guide the selection of regions of interest for further study, which can then be imaged at high sensitivity. In summary, MEZ-XRF enables highly multiplexed imaging with metal tags across multiple biological scales without tissue destruction. This will open new avenues for tissue analysis in health and disease.

Fig. 9: MEZ-XRF scans of three different types of human breast cancer: human epidermal growth factor receptor 2 positive (HER2+), luminal B (LumB), and luminal B HER2 positive (LumB HER2+). In each case, cell types can be identified based on the expression of multiple heavy-metal markers mapped by X-ray fluorescence. The overall composition of cell types determines which cancer treatment will be most beneficial.