Multielement Z-tag imaging by X-ray fluorescence microscopy for next-generation multiplex imaging

Rapid, highly multiplexed, nondestructive imaging that spans the molecular to the supra-cellular scale would be a powerful tool for tissue analysis. However, the physical constraints of established imaging methods limit the simultaneous improvement of these parameters. Whole-organism to atomic-level imaging is possible with tissue-penetrant, picometer-wavelength X-rays. To enable highly multiplexed X-ray imaging, we developed multielement Z-tag X-ray fluorescence (MEZ-XRF) that can operate at kHz speeds when combined with signal amplification by exchange reaction (SABER)-amplified Z-tag reagents. We demonstrated parallel imaging of 20 Z-tag or SABER Z-tag reagents at subcellular resolution in cell lines and multiple human tissues. We benchmarked MEZ-XRF against imaging mass cytometry and demonstrated the nondestructive multiscale repeat imaging capabilities of MEZ-XRF with rapid tissue overview scans, followed by slower, more sensitive imaging of low-abundance markers such as immune checkpoint proteins. The unique multiscale, nondestructive nature of MEZ-XRF, combined with SABER Z-tags for high sensitivity or enhanced speed, enables highly multiplexed bioimaging across biological scales.