Applications of LA-ICP-MS in various fields
Application of the micro-PIXE technique in biomedical research
Preparation of samples for micro-PIXE analysis on biological tissue or individual cells is a demanding process, which dominantly influences the quality of the results. Shock-freezing, cryotome slicing and freeze-drying of the tissues are most frequently used steps for the tissue preparation. However, the removal of water induces morphological alterations at the subcellular level. A need for preserving the sub-cellular morphology motivated the efforts to keep the tissue in a frozen hydrated state during the analysis. First micro-PIXE measurements on frozen hydrated tissues were reported in 2007 by the iThemba group . Several micro-XRF facilities at synchrotrons started to offer such sample handling for external users. We report on instrumental and methodological development of micro-PIXE analysis on frozen hydrated tissue at JSI.
After the incorporation of a multicusp ion source at JSI tandem accelerator, high-energy proton beam brightness is increased for one order of magnitude in comparison with earlier used duoplasmatron ion source . This results in the availability of sub-micrometer beam sizes for micro-PIXE (600 nm) and correspondingly the sub-cellular resolution in the tissue mapping.
Several recent applications of micro-PIXE at JSI are addressing plant physiology , nanomedicine , nanotoxicology , food research  and environmental pollution .
Figure 1. Example: wheat grains exposed to moisture for 2 hours, shock-freezed, freeze-dried, munted on Al holders within two pioloform foils, photographed by fluorescence microscope (Collaboration with K. Vogel Mikuš, Biotechnical faculty, University of Ljubljana).
Figure 2. Parallel acquisition of five spectra in a list mode (OmDaq)
Figure 3. PIXE maps accumulated, OmDAQ, Proton energy 2.5 MeV, overnight
Figure 4. Listmode files processed by GEOPIXE program (C. Ryan)
Figure 5. Zoom on aleurone
Figure 6. Listmode files processed by GEOPIXE program
Figure 7. Last developments: Micro-PIXE on frozen hydrated biological tissue
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Application of synchrotron micro-X-ray fluoresence spectroscopy
A key component of a synchrotron is the storage ring where SR is emitted in a narrow cone in the forward direction, at a tangent to the electron’s orbit. The higher the kinetic energy (i.e. the speed) of the electrons, the narrower the emission cone becomes. The spectrum of the emitted radiation also shifts towards shorter wavelengths as the electron energy increases. In so-called third generation light sources, the storage ring is designed to include special magnetic structures known as insertion devices (undulators and wigglers). Insertion devices generate specially shaped magnetic fields that drive electrons into an oscillating trajectory for linearly polarized light or sometimes a spiral trajectory for circularly polarized light. Each bend acts like a source radiating along the axis of the insertion device, hence the light is very intense and in some cases takes on near-laser-like brightness.
To summarize, synchrotron light has a number of unique properties as:
- high brightness: synchrotron light is hundreds of thousands of times more intense than that from conventional X-ray tubes and is naturally highly collimated;
- wide energy spectrum: synchrotron light is emitted with energies ranging from infrared light to hard X-rays. Furthermore the emitted light is tunable;
- highly polarised: the synchrotron emits highly polarised radiation, which can be linear, circular or elliptical;
- time-structured emission: nano-second long light pulses enable time-resolved studies.
Synchrotron micro-X-ray fluorescence spectroscopy
Synchrotron micro-X-ray spectroscopy (SR-micro-XRF) has become available only recently with the development of the third generation high brilliance synchrotron facilities together with the development of X-ray focusing optics and high-throughput detection systems. SR-micro-XRF is a rapid, non-destructive technique for determination of elements in a wide variety of samples in the ppm or ppb concentration range. The high intensity, linear polarization and natural collimation of synchrotron radiation contribute to the high sensitivity and achievable spatial resolution (below 1 μm) of SR-micro-XRF.
The physical processes are based on the probability that an X-ray photon interacting with an atom will eject a core level electron when the energy of the impinging X-rays is approximately equivalent to or slightly greater that the binding energy of the core level. The de-excitation of the atom via fluorescence X-ray production and the measurement of integrated intensity of the X-ray fluorescence spectrum are then directly related to the element concentration.
XRF techniques therefore offers mapping and quantification, of the elements present in a sample (Figure 1) in a similar way as is possible with non-synchrotron techniques such as EDXRF with all accompanying problems and challenges. The advantage of SR-XRF lies in its sensitivity, due to the high photon flux available, the possibility of beam tunability and weak scattering. Furthermore, the absorption edge of an element is related to the chemical environment of the absorbing atom and its oxidation state. Therefore, by selecting appropriate energies of the incoming X-ray photons, it is possible to generate chemical maps of an element in relation to its oxidation state and chemical bonding. In addition to chemical information based on X-ray Fluorescence (XRF) or micro-X-ray absorption (micro-XAS) spectroscopy, some X-ray microscopes operated at the synchrotron facilities can also provide simultaneously morphological information, through absorption and phase contrast imaging (Figure 2).
Figure 1. Localization of elements in large vacoularized leaf epidermal cell of Thlaspi praecox performed at ID21 beamline, ESRF, Grenoble. A) Stereomicroscope image, B) absorption image, C) Cadmium quantitative distribution map, D) Sulphur quantitative distribution map. Program for quantification analysis was developed by Peter Kump, IJS. (Koren et al. 2013)
Figure 2. Absorption and phase contrast imaging of a large vacoularized leaf epidermal cell of Thlaspi praecox performed at TwinMic beamline, Elettra, Trieste. A) stereomicroscope image, B) absorption image, C) phase contrast image (X-mom), D) phase contrast image (Y-mom).
X-ray microprobes can be roughly divided in soft and hard X-ray microprobes, first operating in the range of 0.12-12 keV and second operating at range over 12 keV. Laboratory and synchrotron-based XRF instrumentation typically uses hard X-rays and only a few soft X-ray XRF instruments have been reported. The use of soft X-rays for XRF analysis has mainly been limited by the low fluorescence yield of low-Z elements and the unavailability of suited detectors and electronics. Such a low-energy XRF system operated in the soft X-ray regime is especially suited for bio-related research as it gives access to the elemental distribution of low-Z elements such as carbon, nitrogen, oxygen, fluorine, iron, zinc, magnesium and other elements with fundamental importance for metabolism in biological systems on the cellular or sub-cellular level.
Figure 3. Twin-Mic beamline at Elettra, Trieste
Figure 4. ID-21 beamline at ESRF, Grenoble
General X-ray microprobe characteristics and set-up
The principal advantage of using synchrotron radiation for XRF analysis is that it allows the spatial resolution of the method to be reduced down to the (sub)micrometer level. There are several reasons why this is possible. As already mentioned, the synchrotron radiation is several orders more intense than X-rays from tube sources. Second, the synchrotron beam is well-collimated, so that the intensity remains high at considerable distances from the source. This means that simple apertures and focusing mirrors can be used to produce small, intense beams. Third, synchrotron radiation is highly linearly polarized which allows background from scattered radiation to be minimized by the geometry of the experiment. Synchrotron micro-XRF (SR-micro-XRF) is complementary to other microanalysis techniques, such as EDX analysis, micro-PIXE, LA-ICP-MS and secondary ion mass spectrometry (SIMS). Each of these techniques is optimized for particular applications, elements, or sample types. The attractiveness of SR-micro-XRF lies in its capability for non-destructive, trace level analysis of a wide range of elements with high spatial resolution. Another advantage is the low power deposition, a particularly important consideration when analyzing volatile-rich specimens or biological materials. For a given fluorescent signal, X-rays deposit between 10-3 and 10-5 times less energy than charged particles.
The SR-micro-XRF probes (see Figures 3 and 4) are therefore particularly well suited for:
- trace element analysis of nanogram samples (e.g., various types of particles, aerosols, and inclusions) and
- characterization of trace element distributions with high spatial resolution (e.g., diffusion profiles, chemical zonation, impurity distribution, and compositional mapping).
Pioneered by Horowitz and Howell (1972), SR-micro-XRF probes are nowadays incorporated in most of the second and third generation synchrotron facilities. Their performance benefits from the latest developments in X-ray optics, detectors and samples environments. SR-micro-XRF instruments rely upon a very specific 45°/45° geometry where the sample is placed at 45° to the incident beam and X-ray detectors are typically placed in the plane of the storage ring and at 90° to the incident beam. The incoming beam is naturally polarized in the horizontal plane, therefore this geometry minimizes the contribution of elastically scattered primary X-rays. Furthermore, the full control of both tunability and spectral bandwidth of the incoming monochromatic radiation minimize the radiation damage without compromising the signal-to noise ratio and allow accurate quantification (within 3–5%). Hard X-rays probe deeper into samples and relax a number of issues associated with attenuation of fluorescence X-rays in the air path and space around the sample.
As depicted in Figure 5, the most advanced hard X-ray microprobes offer a multi-modal microanalysis platform where SR-micro-XRF, SR-micro-XAS and SR-micro-XRD can be combined on the same instrument. All these techniques share the same experimental strategies where a sample is typically mounted on a motorized vertical holder and then translated in the x and y directions through the beam path. Some X-ray microprobe beamlines also have visible, electron or Raman microscope imaging capabilities so that interesting features on a sample can be easily identified and examined by X-ray techniques. Elemental concentrations or chemically specific information is then collected at each pixel to generate corresponding maps. Multi-keV X-ray microscopy often suffers from a low contrast due to a weak absorption of the analyzed samples. Furthermore, the use of absorption contrast can subject the specimen to high radiation doses leading to possible structural changes. Even for radiation of hard materials, many of the samples imaged using fluorescence yield are insufficiently absorbing to provide high contrast images in transmission mode. Therefore accurate morphological localization of trace elements is difficult or even impossible. The development of phase contrast methods fully compatible with detection in the fluorescence yield is therefore essential. Several strategies have been successfully developed and are now routinely used, and include differential phase contrast (DPC) using configured detectors, differential interferential contrast (DIC) with a configured zone-plate or with aperture alignment.
Figure 5. A layout of an X-ray microprobe beamline, providing simultaneous access to various signals. Synchrotron light produced by the undulator is monochromatized by means of silicon crystals and pass further to a Fresnel zone plate and order selecting aperture (OSA), focusing the beam in the level of the sample focal plane. Emitted fluorescence X-rays are then collected by an energy dispersive detector, while transmitted X-ray light can be detected either by photodiode or different charge coupled devices (CCD). A hard X-ray microprobe can operate in air, while soft X-ray microprobes operate in vacuum.
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