German Electron Synchotron - DESY
Radiation damage and structur
DESY is the leading German accelerator center and one of the leading in the world. DESY is a member of the Helmholtz Association and develops, builds and operates large particle accelerators, and uses them to investigate the structure of matter. DESY's combination of photon science and particle physics is unique in Europe. In the field of structural biology DESY operates and provides access to beamline P11 at PETRA III synchrotron source in Hamburg and is funding member of the Center for Structural Systems Biology (CSSB).
Our research in the field of structural biology aims at exploring the new capabilities arising from new brilliant X-ray sources such as PETRA III for structural investigations of metal-organic and biological samples. This includes X-ray crystallography for the investigation of crystallized molecules such as proteins and viruses, and X-ray microscopy for the investigation of aperiodic objects such as cells and tissue. The research further aims at the investigation of dynamical processes, for example charge transfer reactions, and structural changes induced by photo-excitation and studying chemically triggered reactions in microcrystals with X-ray diffraction.
In X-ray crystallography new brilliant synchrotron sources allow structure determination from challenging systems such as membrane proteins, where only very small crystals with sizes down to one micron are available. This not only requires the availability of extremely intense microfocus beamlines such as beamline P11, providing excellent focusing capabilities, but also the development of adequate experimental hardware and sample handling techniques. At beamline P11 we have developed several nano-positioning devices being capable to perform experiments with such (sub)-micrometer sized beams at cryogenic temperatures. For structure determinations from microcrystals we have developed a sample holder from very thin single crystalline silicon, which provides very high transmission of X-rays and does not contribute to any background scattering. This sample holder has been successfully tested with microcrystals and yielded significantly better diffraction data than with any currently available sample holder. In future we plan to perform these micro-focus experiments in an in-vacuum-environment, which will further reduce background scattering caused by air and X-ray window materials. In addition we have successfully applied the method high-pressure freezing wihch
In addition the extremely high photon densities available at newest generation synchrotron sources allow also performing many experiments in a time resolved pump-probe fashion. Here a disturbance of the system is induced e.g. by a laser pulse, and the induced structural changes are followed by X-ray investigation. For this reason a femtosecond pump laser system, which is synchronized to the PETRA III synchrotron, has been installed at beamline P11 and the first successful X-ray spectroscopy experiments have already been carried out with this system. In future we plan to perform time resolved micro-crystallography experiments both with small metal-organic and also larger macromolecular systems on time scales ranging from pico- to milliseconds.
Performing these experiments with microcrystals in a serial way should allow the initiation of chemical reactions not only by laser light, but also by chemicals, for example, with microfluidic mixing. Due to the very small sample dimensions the time for the chemicals to fully penetrate into the microcrystals can be in the microsecond range. In combination with the new X-ray detectors (currently under development at DESY and several other facilities) it will become possible to follow irreversible reactions by taking X-ray snapshots at certain delays after initialization of the reaction.
X-ray microscopy techniques can be divided in two different approaches: full field and scanning techniques. At beamline P11 we currently follow both approaches in order to identify a method or a combination of methods which will be useful to the structural biology user community.
In full field X-ray microscopy, similarly to light microscopy, the entire field of view is illuminated and an objective lens generates a magnified image of the sample on the detector. For weakly absorbing samples such as biological samples, full-field microscopy experiments are typically performed in the so-called “water window” at X-ray energies around 500 eV. Performing these experiments at higher X-ray energies above 2.5 keV has the considerable advantage that the depth of field increases with energy and thicker samples can be investigated. To achieve good contrast here, Zernike phase contrast can be applied by introducing a phase ring into the beam path. At the moment we perform these experiments with biological samples in air at energies around 6 keV yielding a resolution around 50 nm.
In scanning microscopy X-rays are focused into a very small spot and the sample is (raster)-scanned through the beam. In this case the resolution is limited by the convolution of the X-ray beam size and the mechanical stability of the setup. High resolution scanning microscopy requires an extremely high positional stability between the last optical element and the sample. We are currently exploring different ways to achieve highest resolution in 2D and 3D. We have conducted several X-ray fluorescence microscopy and ptychography experiments and were able to continuously improve the resolution of our setup where we have recently achieved sub-10 nm resolution in case of ptychography.
The goal of our research in the field of x-ray microscopy is to develop a highly automated X-ray microscope for routine investigations of biological samples combining both full-field and scanning techniques. All microscopy hardware and software should be very easy to operate - similar to current biological crystallography beamlines. In addition we aim for much faster measurements than at current X-ray microscopes in order to obtain statistically relevant information by the measurement of a large number of samples / specimens.
A major challenge when dealing with (metal-)organic and biological samples and high radiation doses is radiation damage, which strongly limits the information to be obtained from a single sample. Ways to reduce radiation damage are therefore highly desirable and build another part of our research. Conducting experiments at temperatures at helium temperatures, significantly reduces X-ray induced sample alterations. Recent results suggest that the application of high-pressure can also reduce radiation damage, and the first experiments in this direction have already shown very promising results. The goal of this research is to develop and establish the methods and hardware required and finally making these developments available to the user community.
Vartiainen, I. ; Warmer, M. ; Goeries, D. ; et al; Towards tender X-rays with Zernike phase-contrast imaging of biological samples at 50 nm resolution. Journal of synchrotron radiation 21(4), 790 - 794 (2014)
Stellato, F. ; Oberthür, D. ; Liang, M. ; et al; Room-temperature macromolecular serial crystallography using synchrotron radiation.IUCrJ 1(4), 204-212 (2014)
Stachnik, K. ; Meents, A.;Scanning X-ray Microscopy with a Single Photon Counting 2D Detector. Acta physica Polonica / A 125(4), 902 - 906 (2014)
Burkhardt, A. ; Wagner, A. ; Warmer, M. ; et al; Structure determination from a single high-pressure-frozen virus crystal. Acta crystallographica / D 69(2), 308 - 312 (2013)
Samuel, P. P. ; Mondal, K. C. ; Roesky, H. W. ; et al; Synthesis and Characterization of a Two-Coordinate Manganese Complex and its Reaction with Molecular Hydrogen at Room Temperature. Angewandte Chemie / International edition 52(45), 11817 - 11821 (2013)
Mondal, K. C. ; Roesky, H. W. ; Dittrich, B. ; et al; Formation of a 1,4-Diamino-2,3-disila-1,3-butadiene Derivative.Journal of the American Chemical Society 135(43), 15990 - 15993 (2013)