X-ray fluorescence analysis of metal distributions in cryogenic biological samples using large-acceptance-angle SDD detection and continuous scanning at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III.

A new Rococo 2 X-ray fluorescence detector was implemented into the cryogenic sample environment at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III, DESY, Hamburg, Germany. A four sensor-field cloverleaf design is optimized for the investigation of planar samples and operates in a backscattering geometry resulting in a large solid angle of up to 1.1 steradian. The detector, coupled with the Xspress 3 pulse processor, enables measurements at high count rates of up to 106 counts per second per sensor. The measured energy resolution of ∼129 eV (Mn Kα at 10000 counts s-1) is only minimally impaired at the highest count rates. The resulting high detection sensitivity allows for an accurate determination of trace element distributions such as in thin frozen hydrated biological specimens. First proof-of-principle measurements using continuous-movement 2D scans of frozen hydrated HeLa cells as a model system are reported to demonstrate the potential of the new detection system.

Use of small-angle X-ray scattering to resolve intracellular structure changes of Escherichia coli cells induced by antibiotic treatment.

The application of small-angle X-ray scattering (SAXS) to whole Escherichia coli cells is challenging owing to the variety of internal constituents. To resolve their contributions, the outer shape was captured by ultra-small-angle X-ray scattering and combined with the internal structure resolved by SAXS. Building on these data, a model for the major structural components of E. coli was developed. It was possible to deduce information on the occupied volume, occurrence and average size of the most important intracellular constituents: ribosomes, DNA and proteins. E. coli was studied after treatment with three different antibiotic agents (chloramphenicol, tetracycline and rifampicin) and the impact on the intracellular constituents was monitored.

Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action.

Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.