High-resolution X-ray microscopy with a very low dose of X-rays

A team led by the X-ray imaging pioneers at DESY has succeeded in producing high-resolution images of biological structures at very low doses of X-rays. Their new technique was demonstrated by obtaining high-resolution X-ray images of dry biological materials that have not been frozen, coated, or otherwise altered — all with little or no damage to the sample. This procedure uses the type of X-ray interaction commonly used for scanning airport baggage to produce nanometer-resolution images of the material. Using intensely focused high-energy X-rays through a new set of X-ray diffraction lenses, this technique allows imaging of less than 1% of the specimen’s X-ray damage threshold. The experiment involved researchers from the Center for Free Electron Laser Science, Deutsches Elektroniken-Synchrotron, and the Hamburg Center for Ultrafast Imaging in Hamburg, Germany, and Lund University in Sweden.

X-ray beams interact with biological materials in a number of ways, most of which depend on the energy and intensity of the light. At low energies, X-rays are primarily absorbed by sample atoms, which are ionized, causing severe damage to the sample. This image using low-energy X-rays maps the absorption of the sample. At higher energies, absorption is less likely and the material becomes more transparent. In this case, a process called elastic scattering can be used to form the image, in which X-ray photons “bounce” off of matter like billiard balls, without storing any energy. Techniques such as crystallography or ptychography make use of this process. Even so, absorption events still occur, meaning that damage to the sample still occurs. But there’s a third interaction: Compton scattering, in which X-rays leave only a fraction of their energy in the target material. This has been abandoned as a viable X-ray microscopy method, as this interaction only becomes predominant at higher X-ray energies, where previously there were no high-resolution lenses.

The advantage of low doses in samples using high-energy X-rays poses a challenge for making lenses: such X-rays pass through all materials and are hardly refracted or bent as needed for focusing. A new type of diffraction lens, called a multi-layered Laue lens, was developed to address this. Alternating nanometer-thick layers of silicon carbide and tungsten carbide structures are first fabricated layer by layer, which are then used to fabricate holographic optical elements thick enough to focus the beam efficiently.

Using this lens system and a P07 beam line on the PETRA III synchrotron light source on DESY, the team imaged a variety of biological materials by detecting Compton scattering when the sample was rasterized through a focused beam. This scanning mode of the microscope requires a very bright source, which is focused to a point that determines image resolution. PETRA III is the only synchrotron radiation facility bright enough at high energy X-rays to be able to acquire images this way in a reasonable time.

The team used cyanobacterium, diatoms, and even pollen collected directly outside the lab as their samples, managing a resolution of 70 nanometers. Compared to images obtained from similar pollen samples using conventional coherent-scattering imaging methods at 17 keV X-ray energy, the Compton X-ray microscope achieves the same resolution at a dose 2000 times lower.

The results showed that the method could be used to obtain even finer 10nm resolution images before radiation damage became a problem. Future upgrades of synchrotron radiation facilities, such as the PETRA IV facility at DESY, will provide the necessary clarity to achieve this. The method could then be used for imaging whole cells or unsectioned tissues, complementing cryo-electron microscopy and super-resolution optical microscopy, or for tracking nanoparticles within cells, such as for observing drug delivery directly. Compton’s scattering characteristics make this method also ideal for non-biological uses, such as checking the charge and discharge mechanism of a battery.