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 A. 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 single-particle diffraction with x-ray free electron lasers.

Figure 1

(A) A diffraction pattern from E. coli bacteria displayed in a logarithmic scale. (B) The power spectral density of the diffraction pattern, which indicates the resolution extends to 30 nm.

Figure 2

An image reconstructed from Fig. 1. The dense regions inside the bacteria are likely the distribution of proteins labeled with KMnO₄. The semitransparent regions are devoid of yellow fluorescent proteins.

Figure 3

E. coli expressing the indicator protein. Individual bacteria are seen using transmitted light (A and D) and fluorescence (B and E), where the yellow fluorescent protein (green) is seen throughout most of the bacteria except for one small region in each bacterium that is free of fluorescence (arrows), consistent with Fig. 2. C and F show the fluorescent image superimposed on the transmitted light image.