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Biological Imaging by Soft X-Ray Diffraction Microscopy Print

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.

Lenless X-Ray Imaging

Most of us have peered into the eyepiece of an optical microscope, but there is a limit to how small an object we can see, owing to the wavelengths of visible light (about 700 to 400 nm). With wavelengths at least 100 times shorter, x rays offer a path to still finer vision, and scientists have indeed constructed and now regularly use x-ray microscopes. The well-known penetrating power of x rays also means that x-ray microscopes can see into objects opaque to visible light, and tomographic techniques related to those used in CAT scans give access to three-dimensional images. The rub is that lenses that can focus x rays are difficult to make and have a number of limitations. For this reason, researchers around the world interested in both life and physical sciences have been developing a "lensless" alternative to x-ray microscopy.

Lenless imaging uses computational techniques to "reconstruct" an image from diffraction data (the intensities and angles of x rays scattered from the sample rather than transmitted through it). Shapiro et al. have now chimed in with the first lensless imaging of a sample as complex as a eukaryotic cell (a cell with a nucleus), a yeast cell to be exact. Their reconstructed image with a spatial resolution of about 30 nm is two-dimensional. Full three-dimensional images are next on the agenda.

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").

Experimental diffraction data used as input to the difference map algorithm. The continuous diffraction pattern, obtained after approximately 60 s of exposure to the x-ray beam, extends to 10-nm resolution at the edges and spans over six decades of intensity. Insets show magnified views of boxed regions.

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.

(A) Reconstruction of the x-ray wavefield after passage through the cell. The phase information is represented as hue (see color map at left) and the magnitude as brightness. The labels indicate the nucleus (N), vacuole (V), and the cell membrane (M). (B) Scanning transmission x-ray microscope image of the same cell (obtained at the National Synchrotron Light Source with 540-eV x rays and an optic with a Raleigh resolution of 42 nm). This image is for comparison only and was not used in the reconstruction process. (C–D) Two independent reconstructions after the cell was rotated by 3 and 4 degrees with respect to that in (A). The insets show that similar structures on the scale of 30 nm can be clearly seen in each.

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).