|Lensless Imaging of Whole Biological Cells with Soft X-Rays|
|Wednesday, 26 May 2010 00:00|
A team of scientists has used x-ray diffraction microscopy at ALS Beamline 9.0.1 to make images of whole yeast cells, achieving the highest resolution—11 to 13 nanometers (billionths of a meter)—ever obtained with this method for biological specimens. Their success indicates that full 3-D tomography of whole cells at equivalent resolution should soon be possible. The National Center for X-Ray Tomography at ALS Beamline 2.1 images whole, frozen hydrated cells in 3-D (see highlight "Imaging Antifungal Drug Molecules in Action using Soft X-Ray Tomography"). Large numbers of cells can currently be processed in a short time at resolutions of 40 to 60 nanometers, but the ability to increase resolution to the 10-nanometer range would enhance research capabilities in both biology and materials sciences.
This research team had previously imaged frozen hydrated cells at 25 nanometer resolution, but image quality and reconstruction algorithms were affected by ice scattering from outside the cells. This most recent imaging, done at twice the resolution, used freeze-dried yeast cells at room temperature.
While x-rays allow scientists to look deeply into thick specimens, or right through them, imaging with a lens has its own problems. Even the best x-ray microscope lenses (concentric circles of metal known as Fresnel zone plates) cannot focus x-rays with high efficiency. To get an image means using such intense radiation that it more quickly damages biological specimens. At the same time, the geometry of the highest-resolution zone plates makes for an extremely narrow depth of focus.
To get around these barriers, the research team used lensless x-ray diffraction microscopy. The coherent light, light with the same frequency and phase, from Beamline 9.0.1 allowed researchers to produce a high-resolution diffraction pattern from nano-gold labels on non-crystalline structures like the membranes and organelles of a cell. Coherent x-rays are scattered and differentially absorbed by a cell’s internal structures. As the light passes through the cell and reaches the detector, there is no lens either in front of or behind the sample to limit the resolution or efficiency.
The result is a pattern of dark and light specks from the scattered x-rays. A computer uses these patterns to create an image, acting as the “lens” in lensless imaging. Using an algorithm called “Difference Map” the computer converges on the diffraction data through subsequent iterations. Tens of thousands of iterations using the algorithm and manual adjustments of the cell boundaries were needed to yield this experiment’s final image of a pair of yeast cells.
The relationship of a cell’s internal structures can only be determined accurately by full 3-D tomography. Short of true 3-D, it is possible to gain some sense of the arrangement of internal structures by studying images of the structure at various depths, then comparing these with images of the same object made using different techniques.
Additional images of the same freeze-dried cells were made first by scanning transmission x-ray microscopy (STXM), which yielded lower-resolution images but confirmed features in different planes, and then by scanning electron microscopy, which showed surface details in the cell walls that the researchers had labeled with gold position-marking nanoparticles.
Researchers hope to achieve true 3-D images of whole, hydrated, frozen cells at very high resolutions. Rotating the frozen sample in the beam will provide more in-depth information.
Reaping the full benefits of lensless imaging with x-ray diffraction microscopy will require a source of intense coherent light that can yield data at a thousand times the current rate, and a more powerful detector that is currently being constructed. Until then, x-ray diffraction microscopy at Beamline 9.0.1 points the way to what lensless imaging can do.
Research conducted by X. Huang, A.M. Neiman, J. Nelson, J. Steinbrener, and J.J. Turner (Stony Brook University); J. Kirz, D. Shapiro, and S. Marchesini (Berkeley Lab); and C. Jacobsen (Stony Brook University, Northwestern University, and Argonne National Lab).
Research funding: U.S. Department of Energy and the National Institutes for Health. Operation of the ALS and SSRL are supported by the U.S. Department of Energy, Office of Basic Energy Sciences.
Publication about this research: J. Nelson, X. Huang, J. Steinbrener, D. Shapiro, J. Kirz, S. Marchesini, A.M. Neiman, J.J. Turner, and C. Jacobsen, "High-resolution x-ray diffraction microscopy of specifically labeled yeast cells," PNAS 107, 16 (2010). doi: 10.1073/pnas.0910874107
Based on an article by Lawrence Berkeley National Laboratory News Center: http://newscenter.lbl.gov/news-releases/2010/04/27/lensless-imaging/.
ALS Science Highlight #208