Elemental and chemically specific X-ray fluorescence imaging of biological systems.

Abstract

From the perspective of a chemist, biology confers a rich variety of roles on a number of metal ions. It is widely agreed that a large fraction of the genomic output of living things contains metal or metalloid ions, although estimates of this fraction vary widely and depend upon which metal ions are considered.1−3 Moreover, recent reports suggest that, at least in some cases, there are many uncharacterized metalloproteins.4 With inclusion of the s-block metals such as Na, K, Mg, and Ca, the proportion likely approaches 100%; recent estimates from the protein data bank indicate that the prevalence of heavier metal ions of atomic number above 20 within proteins is around 22%,5 with Zn2+ proteins alone constituting about 11%. Living organisms have an inherent and very rich physical structure, with relevant length scales ranging from the nanometer scale for subcellular structure to hundreds of micrometers and above for tissue, organ, or organism-level organization. The ability to derive the spatial distribution of elements on this diversity of length scales is a key to understanding their function. Metals play essential and central roles in the most important and chemically challenging processes required for life, with active site structures and mechanisms that, at the time of their discovery, have usually not yet been duplicated in the chemical laboratory. Furthermore, diseases of metal dysregulation can cause disruption in the distribution of metals.6 For example, Menke’s disease and Occipital Horn Syndrome,7 and Wilson’s disease,8 involve disruption in copper uptake and excretion, respectively, through mutation in the ATP7A and ATP7B Cu transporters.9 The mechanisms of action of toxic elements such as mercury and arsenic are also of interest, as are essential nonmetal trace elements, such as selenium. Likewise, an increasing number of pharmaceuticals include metals or heavier elements; such chemotherapeutic drugs include the platinum derivatives cisplatin and carboplatin,10 some promising new ruthenium drugs,11 and arsenic trioxide, which has been used to treat promyelocytic leukemia.12 Understanding the localization, speciation, and distribution of these at various length scales is of significant interest. A wide variety of heavier elements can be probed by X-ray spectroscopic methods; these are shown graphically in Figure ​Figure1.1. X-ray fluorescence imaging is a powerful technique that can be used to determine elemental and chemical species distributions at a range of spatial resolutions within samples of biological tissues. Most modern applications require the use of synchrotron radiation as a tunable and high spectral brightness source of X-rays. The method uses a microfocused X-ray beam to excite X-ray fluorescence from specific elements within a sample. Because the method depends upon atomic physics, it is highly specific and enables a wide range of chemical elements to be investigated. A significant advantage over more conventional methods is the ability to measure intact biological samples without significant treatment with exogenous reagents. The technique is capable of determining metal and nonmetal distributions on a variety of length scales, with information on chemical speciation also potentially available. Figure ​Figure22 shows examples of rapid-scan X-ray fluorescence imaging at two contrasting length scales: rapid-scan imaging13 of a section of a human brain taken from an individual suffering from multiple sclerosis and showing elemental profiles for Fe, Cu, and Zn;14 and a high-resolution image showing mercury and other elements in a section of retina from a zebrafish larva treated with methylmercury chloride.15 We will discuss both the state of the art in terms of experimental methods and some recent applications of the methods. This Review considers X-ray fluorescence imaging with incident X-ray energies in the hard X-ray regime, which we define as 2 keV and above. We review technologies for producing microfocused X-ray beams and for detecting X-ray fluorescence, as well as methods that confer chemical selectivity or three-dimensional visualization. We discuss applications in key areas with a view to providing examples of how the technique can provide information on biological systems. We also discuss synergy with other methods, which have overlapping or complementary capabilities. Our goal is to provide useful and pertinent information to encourage and enable further use of this powerful method in chemical and biochemical studies of living organisms. Figure 1 Periodic table of the elements showing elements of biological interest that can be probed using X-ray fluorescence imaging. Elements are divided into three categories, those that are physiologically important, those that are pharmacologically active, ...