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, ...

Elemental and Chemically Specific X-ray Fluorescence Imaging of Biological Systems

Spectrum simulation is a useful practical and pedagogical tool. Particularly with complex samples or trace constituents, a simulation can help to understand the limits of the technique and the instrument parameters for the optimal measurement. DTSA-II, software for electron probe microanalysis, provides both easy to use and flexible tools for simulating common and less common sample geometries and materials. Analytical models based on (rhoz) curves provide quick simulations of simple samples. Monte Carlo models based on electron and X-ray transport provide more sophisticated models of arbitrarily complex samples. DTSA-II provides a broad range of simulation tools in a framework with many different interchangeable physical models. In addition, DTSA-II provides tools for visualizing, comparing, manipulating, and quantifying simulated and measured spectra.

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Spectrum simulation in DTSA-II.

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X-ray microscopy station, is reported. The X-ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy-tailored using a collimated plane-grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full-field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X-ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across-absorption-edge imaging, X-ray absorption near-edge structure or low-energy X-ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.

Current status of the TwinMic beamline at Elettra: a soft X-ray transmission and emission microscopy station.

Information on localization of Al in tea leaf tissues is required in order to better understand Al tolerance mechanism in this Al-accumulating plant species. Here, we have used low-energy X-ray fluorescence spectro-microscopy (LEXRF) to study localization of Al and other low Z-elements, namely C, O, Mg, Si and P, in fully developed leaves of the tea plant [Camellia sinensis (L.) O. Kuntze]. Plants were grown from seeds for 3 months in a hydroponic solution, and then exposed to 200 microM AlCl(3) for 2 weeks. Epidermal-mesophyll and xylem phloem regions of 20 microm thick cryo-fixed freeze-dried tea-leaf cross-sections were raster scanned with 1.7 and 2.2 keV excitation energies to reach the Al-K and P-K absorption edges. Al was mainly localized in the cell walls of the leaf epidermal cells, while almost no Al signal was obtained from the leaf symplast. The results suggest that the retention of Al in epidermal leaf apoplast represent the main tolerance mechanism to Al in tea plants. In addition LEXRF proved to be a powerful tool for localization of Al in plant tissues, which can help in our understanding of the processes of Al uptake, transport and tolerance in plants.

Localization of aluminium in tea (Camellia sinensis) leaves using low energy X-ray fluorescence spectro-microscopy.

Novel low-energy X-ray fluorescence (LEXRF) system based on a multiple Si drift detector (SDD) configuration has been developed and implemented in the European TwinMic X-ray microspectroscopy station operating at the Italian synchrotron radiation facility ELETTRA. The setup, hosting up to eight large-area SDDs with specially adapted readout electronics, has demonstrated excellent performance for elemental analysis in the 280–2200 eV photon energy range, which covers the K and L edges of light elements, starting from C. The great advantage is the simultaneous acquisition of LEXRF, absorption and phase contrast maps, providing complementary information on elemental composition and morphology of specimen at submicrometer length scales.

Simultaneous soft X-ray transmission and emission microscopy

Silicon Drift Detectors with integrated FET transistor fabricated at Max-Planck-Institute in Munich in cooperation with PNSensor GmbH are widely used as X-ray sensors in many industrial and scientific applications. In the classical readout scheme, the integrated transistor on the SDD is operated in the source–follower configuration. The signal charge is removed continuously by the detector self-rest mechanism. The method gives very good results at counting rates up to 10 kcps. For higher count rates, the FWHM increases with the growing reset current and a slight shift of the energy peak is observed. The relative large signal rise time can be also a limitation for operation at very high count rates. Alternatively, the SDD can be operated in a Charge Sensitive Amplifier (CSA) configuration. The detector signal charge is integrated on a feedback capacitor across an inverting amplifier with the integrated FET as the input transistor. The signal rise time does not depend on the integrated transistor and can be made very short (e.g. 50 ns). In applications requiring very high counting rates and constant energy resolution, pulsed-reset operation of the SDD is desirable. The signal charge is removed by applying short reset pulses to a reset structure integrated on the detector anode. The combination of the CSA readout scheme and the pulsed-reset method allows the operation at the best energy resolution independent on the count rate.

Optimized readout methods of silicon drift detectors for high-resolution X-ray spectroscopy

To expand the detection efficiency Silicon Drift Detectors (SDDs) with various customized radiation entrance windows, optimized detector areas and geometries have been developed. Optimum values for energy resolution, peak to background ratio (P/B) and high count rate capability support the development. Detailed results on sensors optimized for light element detection down to Boron or even lower will be reported. New developments for detecting medium and high X-ray energies by increasing the effective detector thickness will be presented. Gamma-ray detectors consisting of a SDD coupled to scintillators like CsI(Tl) and LaBr3(Ce) have been examined. Results of the energy resolution for the 137Cs 662 keV line and the light yield (LY) of such detector systems will be reported.

Expanding the detection efficiency of silicon drift detectors

The detection of low-energy protons is a well-known problem in various neutron decay studies. We report first measurements with a silicon drift diode (SDD) using protons with impact energies in the range from 10 to 16 keV. Compared to a standard PIN diode the SDD shows a much improved separation of the proton signal from noise. This detector type initially developed for X-ray spectroscopy will therefore become very useful for proton detection in forthcoming neutron decay experiments. It might also be applied in other projects involving low-energy charged particle detection.

A. Niculae

Papers

Optimized readout methods of silicon drift detectors for high-resolution X-ray spectroscopy

Expanding the detection efficiency of silicon drift detectors

Detection of low-energy protons using a silicon drift detector

F-64 Expanding the Detector Efficiency of Silicon Drift Detectors with Optimized Radiation Entrance Window