- Original article
- Open Access
Low-dose three-dimensional hard x-ray imaging of bacterial cells
© Bartels et al.; licensee Springer. 2012
Received: 15 July 2012
Accepted: 2 October 2012
Published: 30 November 2012
We have imaged the three-dimensional density distribution of unstained and unsliced, freeze-dried cells of the gram-positive bacterium Deinococcus radiodurans by tomographic x-ray propagation microscopy, i.e. projection tomography with phase contrast formation by free space propagation. The work extends previous x-ray imaging of biological cells in the simple in-line holography geometry to full three-dimensional reconstruction, based on a fast iterative phase reconstruction algorithm which circumvents the usual twin-image problem. The sample is illuminated by the highly curved wave fronts emitted from a virtual quasi-point source with 10 nm cross section, realized by two crossed x-ray waveguides. The experimental scheme allows for a particularly dose efficient determination of the 3D density distribution in the cellular structure.
Optical nanoscopy based on visible light fluorescence is becoming an important tool for three-dimensional imaging of biological cells at the nanoscale. However, not all biological problems can be addressed based on the distribution of fluorescence markers. In many instances additional and complementary contrast mechanisms are needed. The mass density distribution within native unstained and unsliced biological cells and tissues is such a case in point. To this end, coherent x-ray imaging and tomography offers a unique potential for quantitative three-dimensional (3D) density determination at scalable resolution (Cloetens et al. 2006; Huang et al. 2009; Lima et al. 2009; Nishino et al. 2009; Shapiro et al. 2005; Song et al. 2008). The distribution of density contrast in the sample is obtained from quantitative phase reconstruction schemes ((Nugent 2010; Paganin 2006), and ref. therein), along with the reconstructed volumes, shapes and topologies. The density based contrast mechanism equips biophysical research with a non-destructive ’ultra-centrifuge’ at the organelle and supra-molecular level. In other words, quantitative mass density measurements and density based discrimination, which is important in many biological applications, but typically associated with destructive separation of components, can be performed non-destructively. Important problems associated with sub-cellular architecture, as for example DNA compactification can thus be addressed. However, for biological samples the applied dose is a crucial parameter in view of structural changes and radiation damage during the imaging process, inhibiting live cell imaging and creating a need for fixation. Moreover, radiation damage of samples is considered to be the ultimate limitation in x-ray microscopy, if resolution due to the characteristics of sources, optical components, reconstruction algorithms and detection is successfully scaled down (Howells et al. 2009). Therefore, minimizing the dose for a given image resolution and contrast is a primary challenge for x-ray optics.
Here we present a 3D tomographic reconstruction of unstained freeze-dried cells of the gram-positive bacterium Deinococcus radiodurans using hard x-rays (13.8 keV photon energy). The total dose applied during the tomographic scan was about 1.6·105 Gray ([GY=J/kg]), corresponding to 1.9·103 Gy per projection, which is several orders of magnitude below the values reported in previous x-ray coherent diffractive imaging (CDI) studies of D. radiodurans (Lima et al. 2009; Giewekemeyer et al. 2010; Wilke et al. 2012). Despite the low dose, phase reconstruction based on recorded holographic intensities was not hampered by noise, primarily due to two reasons: (i) the use of a highly coherent beam with curved wavefronts (Salditt et al. 2011), emitted by a quasi-point source with 10 nm cross section, realized by a 2D x-ray waveguide system (Krüger et al. 2010 2012), and (ii) a robust and quickly converging iterative reconstruction scheme which takes photon noise effects into account quantitatively (Giewekemeyer et al. 2011). The electron density distribution and an effective mass density distribution of the cellular structure was extracted quantitatively from the reconstructed 3D phase information. Density contrast may help to understand the bacterium’s extraordinary resistance to high doses of ionizing radiation based on the structural arrangement of its nucleoid (Eltsov and Dubochet 2006a 2006b; Minskey et al. 2006). Coherent x-ray imaging was previously used to derive the projected electron density of D. radiodurans (Giewekemeyer et al. 2010), providing thus a contrast mechanism complementary to electron microscopy studies (Eltsov and Dubochet 2005; Levin-Zaidman et al. 2003). The data was recorded by scanning a micron-sized x-ray beam over the sample, followed by reconstruction of the far-field diffraction patterns, yielding the super-resolution real space projection image. The reconstruction algorithm used was based on the a priori knowledge of partial overlap between adjacent images, reducing the number of unknown variables, an approach denoted by ptychography (Dierolf et al. (2010)). In order to obtain the locally resolved electron and mass density rather than just projected values, we here extend this work from 2D to 3D.
However, instead of ptychographic tomography which involves scanning two translations and one rotation with correspondingly large overhead time (Dierolf et al. 2010; Wilke et al. 2012), in this work we use x-ray propagation microscopy, i.e. projection imaging with contrast formation by free space propagation (Cloetens et al. 1999; Wilkins et al. 1996). In our view, replacing the (Fraunhofer) far-field recording with a magnified (Fresnel) near-field setting (Giewekemeyer et al. 2011, Mokso et al. 2007) has the following advantages: (i) The near-field pattern directly represents location and shape of the object, so that object support and position in the beam can be readily located, giving useful a priori knowledge. (ii) Current detector technology is better exploited due to homogenous signal level. Furthermore, (iii) a high dose efficiency is gained by interference of the weak diffracted wave with the much stronger primary wave as demonstrated experimentally in this work. Finally, (iv) in view of 3D imaging, a full field approach based on scanning only one degree of freedom (rotation axis) produces less overhead time in data acquisition and imposes less restrictive requirements on mechanical accuracy and longterm vibrational stability. However, shaping a highly coherent beam with curved wavefronts is a major challenge in propagation imaging. By using x-ray waveguides the well-known problems of empty beam correction and geometrical distortions associated with hard x-ray focusing optics (Mokso et al. 2007) can be solved (Giewekemeyer et al. 2011).
In summary, we have used phase contrast projection tomography to compute the 3D density distribution in bacterial cells, showing characteristic density maxima which we attribute to DNA rich regions. The imaging and reconstruction method based on near-field propagation or in-line holographic recording is particularly dose efficient. Application of the method to multi-cellular organisms and tissues would allow a large field of view in all spatial dimensions at sub-cellular resolution without the need of staining or slicing. The ability to easily adapt the field of view and resolution by changing the defocus distance could be used as a zoom and advantageously combined with region-of-interest (local) tomography, which however would imply generalizations of the phase reconstruction algorithms. With further technological progress (waveguide transmission, vibrational stability), resolution can be increased up to the theoretical limit set by the extension of the quasi-point source used for illumination, which was 10 nm in the present case. The result of the 2D test structure, for which a waveguide with particularly high flux was available, already demonstrates that the waveguide setup is in principle well suited for imaging at a resolution below 50 nm, a field of view in the range of several microns up to a few hundreds of microns, and one second accumulation time, paving the way for relatively fast and dose efficient nanotomography applications.
We thank HASYLAB for beamtime and the PETRA III team for excellent beam conditions. Funding by the DFG collaborative research center SFB 755 Nanoscale Photonic Imaging and the German Ministry of Education and Research (Grant No. 05K10MGA) is gratefully acknowledged.
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