With a combined X-ray technique, scientists have traced the players for tuberculosis drugs within cells with very high accuracy. The method combines two sophisticated X-ray measurements and can find minutes of different metals in biological samples at a very high resolution, as reported by a team around a scientist DESY Karolina Stachnik in the journal. Science Reports. To illustrate its flexibility, the researchers also used the method of combining the calcium content of the human bone, which can benefit from osteoporosis research.
Metals play a key role in numerous biological processes, from the oxygen behavior in our red blood cells and from the mineralization of bones to the accumulation of harmful metal in nerve cells as seen in diseases such as Alzheimer’s. “
Karolina Stachnik, scientist, Center for Electronics Laser Electron-Electron CFEL at DESY
High energy X-rays light up metal in fluorescence, which is very sensitive even for small amounts. “However, X-ray fluorescence measurements do not usually show a basic cell structure, for example,” said DESY scientist Alke Meents who led the research. “If you want to find the metals directly within your example, you must combine the measurements with an imaging technique.” The infrastructure includes data of cell morphology not visible under an optical microscope.
Because biological samples, such as cells, are very sensitive to X-ray radiation, it is highly beneficial to imaging its structure simultaneously with the fluorescence analysis. For this reason, the team of fluorescence measurements combined with an imaging method called ptychography. “A ptychographic microscope is like taking a panoramic image,” explains Stachnik. “An extended specimen such as a biological cell is the scanning of raster with a small X-ray beam that produces many overlapping images of parts of the sample. These overlapping images are then sewn together.”
The application method operates without lenses between the sample and the detector, resulting in X-ray diffraction patterns being recorded on the detector. Each of these patterns is known on the spatial structure of the particular part of the sample, which can be calculated from the pattern. “This results in a full quantitative optical density map of the specimen,” explains Stachnik. “Through this complex process, ptychography provides spatial secrets over normal limits of X-ray optics.”
Thanks to its scanning nature, ptychography can be combined with the simultaneous acquisition of X-ray fluorescence measurements that provide a unique fingerprint of the sample-based elements. In this way, a photograph of the morphology of the sample can be obtained by ptychography to overlay an element map. “Due to the concurrent combination of these two complementary imaging methods, therefore, artificial correlations of artefacts with trace elements of a very exemplary structure can be summarized”.
It is a fundamental prerequisite that the X-rays are one color (monochromatic, all of which have the same wavelength) and that they open in a stage (progressive) such as a laser. “Meents are only consistent with modern synchrotron light sources such as PETRA III DESY” that only bright cohesive monochromatic rays with high energy enough to allow metals like iron fluoreses.
To test the method, researchers DESY joined the Ulrich Schaible group from the Borstel Research Center to investigate localization and concentration of nanocariers for tuberculosis drugs in macrophages, filter cells of the immune system. “Usually, macrophages destroy pathogens such as viruses and bacteria. Unfortunately, tuberculosis bacteria have managed to avoid destruction and hide them inside the microphages, instead of using them to grow”, Schaible says. “To prevent effective treatment, antibiotics must be achieved in the niches of bacteria in macrophages to be effective.”
A new “Trojan Horse” strategy uses nanometer iron containers to deliver antibiotics directly into the cells. These containers are hollow, filled with antibiotics and measure less than 20 nanometers in diameter (the nanometer is million millimeters). “The containers are swallowed in Macrophages, and when they are inside the cell, the cage walls slowly dissolve due to the need for iron bacteria.
In order to assess the effectiveness of this strategy, the team investigated macroparines fed by iron containers. Using a specially developed scanning phase at the bio-imaging line and P11 diffraction of X-ray source DESY PETRA III, the researchers could capture ptychographic and fluorescence images of 14 cells with sub-acetic resolution and d ‘ they identified 22 agglomerations of nano-owners.
In the second application the researchers collaborated with the Björn Busse group from the University Medical Center Hamburg-Eppendorf (UKE) and analyzed the calcium content in a sample of human bone. “Calcium is a major feature of our bones”, explains co-author Katharina Jähn from the Busse group. “However, in times of high calcium requirement, the body dissolves it from the bones that are to be used elsewhere. These can lead to osteoporosis and other age-related processes, which affect t nearly a quarter of all women over 50 are in Germany. “
Experimental mineralization of bone is usually carried out on small bone slices. “However, only the total calcium content is usually mapped in this way,” says Stachnik. “For correct measurement of the calcium concentration, one must be corrected for the thickness of the sample that is often different.” The team used a ptychographic image found at the same time to eliminate the mass-thickness distortion from the calcium distribution map. “With this approach we were able to observe calcium content that was lower locally at certain points in the bone, which helps to better understand the process of bone disorders and to affect bone mineral changes in patients. “, Stachnik emphasizes.
To further improve the method, the researchers have begun to extend the analysis to three dimensional measurements. “The experimental setup is currently being extended to obtain 3D-tomographic datasets at P11 beamline,” says Meents. “As many synchrotrons are being upgraded to produce brighter X-rays, we expect the method to increase throughput and become the normal function of these facilities.”
This research involved the Borstel Research Center, the Paul Scherrer Institute in Switzerland, the Karlsruhe Institute of Technology, the University Medical Center Hamburg-Eppendorf and DESY.
DESY is one of the largest particle accelerator centers in the world and investigates the structure and function of the material – from the interaction of tiny basic particles to the transport of novel nanomaterials and vital biomolecules to the great mystery of the world. Accelerators and particle detectors are unique research tools that develop and build DESY at their locations in Hamburg and Zeuthen. They generate the most intense X-ray radiation in the world, accelerate particles to record energy and open new windows on the globe. DESY is a member of the Helmholtz Association, the largest scientific association in Germany, and receives funding from the German Federal Ministry of Education and Research (BMBF) (90 percent) and from German federal states Hamburg and Brandenburg (10 percent) .
Deutsches Elektronen-Synchrotron DESY
Reference to journal:
Stachnik, K., et al. (2020) Mixed X-ray imaging of treated microphages and calcium distribution in a bone marrow matrix. Science Reports. doi.org/10.1038/s41598-020-58318-7.