| AVS 60th International Symposium and Exhibition | |
| Nanoparticle-Liquid Interfaces Focus Topic | Friday Sessions |
| Session NL+AS+BI+SA-FrM |
| Session: | Emerging Methods to Identify and Measure Nanomaterials in Biological Environments |
| Presenter: | C.A. Larabell, University of California, San Francisco |
| Correspondent: | Click to Email |
SXT is similar in concept to the well-established medical diagnostic technique, computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. We examine whole, hydrated cells (between 10-15 µm thick), eliminating the need for time-consuming and potentially artifact-inducing embedding and sectioning procedures. Cells are rapidly frozen then imaged using photons with energies between the K shell absorption edges of carbon (284 eV, λ=4.4 nm) and oxygen (543 eV, λ=2.3 nm). In this energy range, photons readily penetrate the aqueous environment while encountering significant absorption from carbon- and nitrogen-containing organic material. Consequently organic material absorbs approximately an order of magnitude more strongly than water, producing a quantifiable natural contrast image of cellular structures. By collecting images from multiple angles through 360 degrees of rotation, SXT reconstructions yield information at isotropic resolution.
Images are formed using unique optics called zone plates (ZP). An X-ray ZP optic consists of a number of concentric nanostructured metal rings, or zones, formed on a thin X-ray transmissive silicon nitride membrane. The width of the outermost ring determines the spatial resolution of the ZP lens, whereas the thickness of the rings determines the focusing efficiency. In our microscope, we use a condenser ZP lens with an overall diameter of 1 cm and an outer zone width of 50 nm. The high-resolution objective ZP lens has a diameter of 63 µm, 618 zones, a focal length of 650 µm at 2.4 nm wavelength, and an outer zone width of 50 nm.
Because SXT is fast (~ 5 min per tomographic data set), we can examine large numbers of cells. Since organic material absorbs approximately an order of magnitude more strongly than water, a unique and quantifiable natural contrast image of cellular structures is generated. X-ray absorption follows Beer’s Law, therefore the absorption of photons is linear and a function of the biochemical composition at each point in the cell. As a result, a linear absorption coefficient (LAC) value of each voxel can be calculated. For example, lipid drops with high concentrations of carbon are more highly absorbing (LAC=0.7 mm-1) than fluid-filled vesicles (LAC=0.2 mm-1). We can determine the position of specific molecules by overlaying fluorescence microscopy signals on cell structures obtained with x-ray imaging. In addition, we can directly determine the locations and numbers of metal probes throughout the cell.