Double X-Ray Vision

Two agglomerates of antibiotic-loaded iron nanocontainers in a macrophage. (Source: Stachnik et al., NPG, CC BY 4.0)

With an advanced X-ray combi­nation technique, scientists have traced nano­carriers for tuberculosis drugs within cells with very high precision. The method developed by a team around DESY scientist Karolina Stachnik combines two sophis­ticated scanning X-ray measurements and can locate minute amounts of various metals in biological samples at very high resolution. To illustrate its versatility, the researchers have also used the combi­nation method to map the calcium content in human bone, an analysis that can benefit osteo­porosis research.

“Metals play key roles in numerous biological processes, from the oxygen transport in our red blood cells and the minerali­sation of bones to the detrimental accu­mulation of metals in nerve cells as seen in diseases like Alzheimer’s,” explains Stachnik who works in the Center for Free-Electron Laser Science CFEL at DESY. High-energy X-rays make metals light up in fluores­cence, a method that is very sensitive even to tiny amounts. “However, the X-ray fluorescence measurements usually do not show the ultra­structure of a cell, for example,” says DESY scientist Alke Meents who led the research. “If you want to exactly locate the metals within your sample, you have to combine the measurements with an imaging technique.” The ultra­structure comprises the details of the cell morpho­logy that are not visible under an optical micro­scope.

As biological samples, such as cells, are very sensitive to X-ray radiation, it is highly beneficial to image their structure simul­taneously to the fluorescence analysis. For this reason, the team combined the fluores­cence measurements with an imaging method known as ptychography. “A ptycho­graphic microscope is fairly similar to taking a panorama image,” explains Stachnik. “An extended specimen like a biological cell is raster scanned with a small coherent X-ray beam that produces many over­lapping images of parts of the sample. These overlapping images are then stitched together afterwards.”

The applied method works without any lenses between the sample and the detector, and as a consequence X-ray diffraction patterns are recorded on the detector. Each of these patterns contains infor­mation on the spatial structure of the respective part of the sample, which can be calculated from the pattern. “This finally results in a fully quanti­tative optical density map of the specimen”, explains Stachnik. “Via this complex process, ptycho­graphy provides spatial resolutions beyond the usual limits of X-ray optics.”

Thanks to its scanning nature, ptychography can be combined with simultaneous acquisition of X-ray fluorescence measurements that provide a unique finger­print of the sample-constituting elements. In this way, a photograph of the sample’s morpho­logy obtained by ptychography can be overlaid with an element map. “The concurrent combi­nation of these two complementary imaging methods enables therefore artefact-free corre­lations of trace elements with the highly resolved specimen’s structure,” summarises Meents.

A fundamental prerequisite is that the X-rays are of a single colour only and that they oscillate in step like in a laser. “Suffi­ciently bright coherent mono­chromatic X-rays with energies high enough to let metals like iron fluoresce have only become available at modern synchrotron light sources like DESY’s PETRA III”, says Meents.

To test the method, the DESY researchers teamed up with the group of Ulrich Schaible from the Research Center Borstel to investigate localisation and concen­tration of nanocarriers for tuberculosis drugs within macro­phages, the scavenger cells of the immune system. “Usually, macrophages destroy pathogens like viruses and bacteria. Unfor­tunately, tuberculosis bacteria have managed to evade destruction and hide inside the macrophages instead, even using them to grow”, says Schaible. “As a barrier for effective treatment, the bacteria’s niches within macrophages need to be reached by antibiotics to be efficient.”

A new “Trojan Horse” strategy uses nanometre-sized iron containers to deliver antibiotics directly into the cells. These containers are hollow, filled with antibiotics and measure less than 20 nanometres in diameter. “Macro­phages swallow the containers, and once they are inside the cell, the iron walls of the cages slowly dissolve due to the need of the bacteria for iron. Eventually, the antibiotics are released and kill the bacteria”, explains Schaible. To evaluate the efficacy of this strategy, the team inves­tigated macrophages that had been fed iron containers. Using a specially developed scanning stage at the bio-imaging and diffraction beamline P11 of DESY’s X-ray source PETRA III, the researchers could capture ptycho­graphic and fluorescence images of 14 cells with sub­cellular resolution and identified a total of 22 agglomerates of nanocontainers within them.

In a second application the researchers teamed up with the group of Björn Busse from the University Medical Center Hamburg-Eppen­dorf (UKE) and analysed the calcium content in a sample of human bone. “Calcium is a key element that makes our bones strong”, explains Katharina Jähn from Busse’s group. “However, in times of high calcium requirement, the body dissolves it from the bones to be used elsewhere. These and other age-related processes can lead to osteo­porosis, affecting nearly a quarter of all women at ages over 50 years in Germany.”

Experi­mental research on bone minerali­sation is usually performed on small slices of bone. “However, only the total content of calcium is usually mapped this way,” says Stachnik. “To get a true measure of the calcium concentration, one has to correct for the often varying thickness of the sample.” The team used a simul­taneously obtained ptycho­graphic image to remove the mass-thickness distortion from the calcium distri­bution map. “With this approach we were able to observe a locally lower calcium content at certain points in the bone, which helps to better understand the process of bone disorders and to quantify the effect of bone mineralisation changes in patients”, emphasises Stachnik.

To improve the method even further, the researchers have started to extend the analysis to three-dimensional measurements. “The experi­mental setup is currently being extended to allow acquisition of 3D-tomo­graphic datasets at beamline P11,” says Meents. “With many synchrotrons being upgraded to produce even brighter X-rays, we expect the method to increase throughput and to become a routine appli­cation at these facilities.” (Source: DESY)

Reference: K. Stachnik et al.: Multimodal X-ray imaging of nanocontainer-treated macrophages and calcium distribution in the perilacunar bone matrix, Sci. Rep. 10, 1784 (2020); DOI: 10.1038/s41598-020-58318-7

Link: Coherent Imaging, Center for Free-Electron Laser Science CFEL, DESY, Hamburg, Germany

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