A comparison of absorption and phase contrast for X-ray imaging of biological cells.

X-ray imaging allows biological cells to be examined at a higher resolution than possible with visible light and without some of the preparation difficulties associated with electron microscopy of thick samples. The most used and developed technique is absorption contrast imaging in the water window which exploits the contrast between carbon and oxygen at an energy of around 500 eV. A variety of phase contrast techniques are also being developed. In general these operate at a higher energy, enabling thicker cells to be examined and, in some cases, can be combined with X-ray fluorescence imaging to locate specific metals. The various methods are based on the differences between the complex refractive indices of the cellular components and the surrounding cytosol or nucleosol, the fluids present in the cellular cytoplasm and nucleus. The refractive indices can be calculated from the atomic composition and density of the components. These in turn can be obtained from published measurements using techniques such as chemical analysis, scanning electron microscopy and X-ray imaging at selected energies. As examples, the refractive indices of heterochromatin, inner mitochondrial membranes, the neutral core of lipid droplets, starch granules, cytosol and nucleosol are calculated. The refractive index calculations enable the required doses and fluences to be obtained to provide images with sufficient statistical significance, for X-ray energies between 200 and 4000 eV. The statistical significance (e.g. the Rose criterion) for various requirements is discussed. The calculations reveal why some cellular components are more visible by absorption contrast and why much greater exposure times are required to see some cellular components. A comparison of phase contrast as a function of photon energy with absorption contrast in the water window is provided and it is shown that much higher doses are generally required for the phase contrast measurements. This particularly applies to those components with a high carbon content but with a mass density similar to the surrounding cytosol or nucleosol. The results provide guidance for the most appropriate conditions for X-ray imaging of individual cellular components within cells of various thicknesses.