Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction

Abstract

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining singleparticle diffraction with x-ray free electron lasers. Single-particle diffraction is a methodology of extending crystallography to determine the 2D and 3D structures of nano crystals and noncrystalline samples by using coherent x-rays and electrons (1-6). In this approach, coherent diffraction patterns are recorded and then converted directly to highresolution images by using the oversampling phasing method (7). Due to the lack of multiple copies of the object, which constructively reinforce due to Bragg diffraction, the diffraction patterns are usually weak and continuous. This illustrates why the experiments with high Z scatters, such as those made of Au and Ni, are simpler to carry out (2-4). However, some of the most important potential applications of single-particle diffraction lie in biological materials (8, 9). Here we report the first successful experiment, to our knowledge, of imaging Escherichia coli bacteria at 30-nm resolution by using single-particle x-ray diffraction. The images show the distribution of manganese-tagged proteins, which is consistent with results from fluorescence microscopy. MethodsExperimental Setup. The coherent diffraction experiment was conducted on an undulator beamline at SPring-8 (10). Because single-particle x-ray diffraction requires good spatial (i.e., beam divergence) and moderate temporal coherence (i.e., energy spread) (4), we achieved the beam divergence of Ϸ6 ϫ 10 Ϫ6 rad by setting a 150-m pinhole at a distance of 27 m upstream of the experimental instrument, and the energy spread of 0.7 eV (1 eV ϭ 1.602 ϫ 10 Ϫ19 J) by using a Si (1,1,1) double crystal (10). To make a small clean beam, a 20-m pinhole and a corner were placed at a distance of 25.4 and 12.7 mm upstream of the sample. The pinhole and corner combination created three very clean quadrants on a charge-coupled device (CCD) detector for recording weak diffraction patterns. The intensity lost in the noisy fourth quadrant was recover...

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