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New bioimaging technique exploits high-energy X-ray fluorescence

A new biological imaging technique combining molecular tagging with X-ray fluorescence microscopy enables ultrafast, non-destructive and multiplexed targeted molecular contrast for X-ray bioimaging, spanning the tissue- to subcellular- resolution range. The technique could open new avenues for tissue analysis.

In order to diagnose diseases such as cancer, powerful biomedical imagery is needed to analyse tissue samples across different spatial scales down to the subcellular level, ideally in a non-destructive manner in order to allow repetitive imaging. However, established imaging methods are limited and often maximise one or two parameters at the expense of others. For instance, electron microscopy can image at sub-nanometre resolution, but at low speed, low throughput and with limited ability to multiplex (i.e., see several different key markers of the disease in parallel). On the other hand,

such multiplexing is possible with optical fluorescence imaging, where certain molecules in a sample are tagged with molecular fluorophores, which fluoresce when illuminated with light of a specific wavelength. However, spectral overlap is constrained by the number of fluorescent tags that can be imaged simultaneously, restricting the feasible number to approximately four or five. Imaging mass cytometry (IMC) allows for imaging of 40 or more tags at cellular (micron) resolution by tagging specific molecules with antibodies bound with heavy metal isotopes, which are scanned with a laser, although it is generally limited to 2D and is a destructive technique, precluding any subsequent analyses on the same tissue. Moreover, efforts were made to recreate 3D imagery from serially sectioned 2D IMC images; however, this procedure proved to be time-consuming, requiring a week for the analysis of a human breast cancer sample [1].

A new technique called multielement Z-tag imaging by X-ray fluorescence (MEZ-XRF) was developed with the aim to overcome such issues. X-rays are ideal for repetitive imaging of bulk or sectioned biological samples across a range of spatial resolutions. Furthermore, X-ray microscopy can be combined with spectroscopic methods such as element-sensitive XRF, exploiting the fact that elements with different atomic numbers fluoresce with signature wavelength emissions when excited by an X-ray beam. Tissue samples of human breast tumours, tonsils and appendixes were stained with up to 20 heavy metal isotopes (mainly lanthanides) called Z-tags and

Fig. 8: Principle of MEZ-XRF, involving (1) staining of biological

samples with Z-tagged affinity reagents, (2) raster scanning of a focused X-ray

beam over the stained sample and collecting emission spectra for each pixel,

(3) deconvoluting spectra into multichannel images and (4) analysing images.

Resolution is determined by the focus and step size of the raster X-ray beam. Repeat imaging at different resolutions

enables multiscale MEZ-XRF.