|Biological Imaging by Soft X-Ray Diffraction Microscopy|
Electron and x-ray microscopes are a valuable tool for both the life and materials sciences, but they are limited in their ability to image with nanometer-scale resolution in three dimensions nonperiodic objects that are several microns in size. To fill this gap, the technique of coherent x-ray diffraction imaging now under development takes advantage of the penetrating power of x rays while simultaneously removing the limitations imposed by lens-based optical systems. Researchers from Stony Brook University, in collaboration with scientists at the ALS and Cornell University, have taken a large step in this direction by using a lensless x-ray diffraction microscope to image a freeze-dried yeast cell to better than 30-nm resolution. Images were made at several angular orientations of the cell.
Electron microscopes are limited to biological specimens no thicker than a bacterium, and the technological challenges involved with making high-efficiency diffractive x-ray optics limits the capabilities of current lens-based x-ray microscopes. Coherent x-ray diffraction imaging, also known as x-ray diffraction microscopy (XDM), uses only the scattered intensities in the far field to recover the structure of the scattering object. If the object is nonperiodic, then the far-field intensity pattern is continuous and can be sampled finely enough that the phase, which is lost when intensity measurements are made, can be recovered by iterative computational methods (see previous highlight, "Demonstration of Coherent X-Ray Diffraction Imaging").
In XDM, the action of a lens is replaced by a computational process that recovers the far-field phase information through the iterative application of constraints in both image and diffraction space. These constraints describe a priori knowledge that we have about the object and typically take the form of a support constraint (e.g., a requirement that the sample be finite and isolated) and a Fourier modulus constraint (a requirement that the calculated diffraction intensities match the measurements). This method of imaging a non-periodic object by phasing its continuous diffraction pattern was first suggested by David Sayre (then at IBM T.J. Watson Research Center) and first demonstrated with x rays by John Miao (then at Stony Brook University) and others in experiments at the National Synchrotron Light Source.
The current work represents the first application of XDM to image an object as complex as a eukaryotic cell. The diffraction microscope, developed by researchers at Stony Brook and now stationed at ALS Beamline 9.0.1, is capable of collecting three-dimensional diffraction data sets from dry or frozen hydrated specimens to a scattering angle that corresponds to a reconstructed half-period pixel size of 6 nm for 750-eV x rays. The diffraction pattern that the researchers used for their image reconstruction is a subset (1200 x 1200 pixels) of the full CCD recording and extends to 10-nm resolution. Approximately 400 intensities at low spatial frequencies are missing because of the need to block the brightest part of the pattern to prevent damaging the detector.
The phasing algorithm, developed at Cornell and known as the difference map, controls these intensities and calculates the missing phases. The reconstructed image contains both magnitude and phase information (not the same as the diffraction phases calculated by the algorithm) about the scattered wavefield. An averaging technique was developed to minimize the effects of noise on the reconstruction. Similar reconstructions were obtained from eight angular orientations of the cell at 1° rotation intervals. The good agreement between the independently recovered structures provides confidence in the fidelity of the reconstructed images, and a comparison of adjacent reconstructions indicates a spatial resolution of better than 30 nm. This estimate is supported by an analysis of an effective modulation transfer function for the microscope.
Experiments aimed at imaging a frozen hydrated cell in three dimensions are ongoing and will allow us to test our calculations that show the radiation-damage-limited resolution of XDM to be 10 nm for biological specimens.
Research conducted by D. Shapiro, E. Lima, H. Miao, A.M. Neiman, and D. Sayre (Stony Brook University); P. Thibault and V. Elser (Cornell University); T. Beetz and C. Jacobsen (Stony Brook University and Brookhaven National Laboratory); M. Howells (ALS); and J. Kirz (Stony Brook University and ALS).
Research funding: National Institutes of Health, National Science Foundation, and U.S. Department of Energy, Office of Basic Energy Sciences (BES). Operation of the ALS is supported by BES.
Publication about this research: D. Shapiro, P. Thibault, T. Beetz, V. Elser, M. Howells, C. Jacobsen, J. Kirz, E. Lima, H. Miao, A.M. Neiman, and D. Sayre, "Biological imaging by soft x-ray diffraction microscopy," Proc. Nat. Acad. Sci. USA 102, 15343 (2005).