The proton or (nuclear) microprobe quantitatively maps simultaneous element distributions within microscopic regions of a sample via the use of particle (proton)-induced X-ray emission (PIXE), mass by scanning transmission ion microscopy (STIM) and low Z elements (Z < 12) through nuclear reactions or scattering. This technique provides spatial resolution down to 1 |lm in a sample, and has been used to successfully perform quantitative analyses of element distributions in individual cells and thin-tissue sections for toxicology, physiology, structural biology, and biochemistry applications. It has also been used to analyze native electrophoretic gel blots for bound ligands, with same sample analysis of protein mass for stoichiometric relationships.58-62 For our application to bioweapon surrogate spore samples, both bulk and individual particulate analysis was performed.
The microprobe source, accelerator and beam lines occupy an area of 3 X 20 meters. Starting at one end, Hv or O- ions are produced using a gas-fed ion source. The Hv or O- ions are then mass/energy-analyzed by the low-energy injection magnet and injected into a National Electrostatics Corporation 5SDH-2 tandem accelerator. The maximum operating voltage of the accelerator is 1.7 MeV, but normal operating voltage is 1.5 MeV, to produce ~3.0 MeV protons or 6.0 MeV O3+. The accelerated ions are collimated and focused on samples contained in the sample chamber at the end of the beam line.
With PIXE, a beam of MeV energy protons is used to eject inner shell electrons from specimen atoms. When outer shell electrons fill the resulting vacancies, characteristic X-rays whose energies identify the particular type of atom are emitted. X-rays from multiple elements (Z > 11) are simultaneously detected. PIXE quantifies elements (Z > Na) by counting elementally characteristic X-rays emitted from a defined spatial region when the sample is struck by a known amount of accelerated protons. PIXE is analytically quantitative with elemental sensitivities that can approach 0.1 mg/kg.63
Microbeam PIXE (| -PIXE) utilizes a focused beam that is scanned across the sample. X-rays from multiple elements are simultaneously detected and recorded as a function of position as the beam is rastered across the sample. The proton beam can be focused to under 1 | m in diameter for investigating detailed element distributions in single cells, isolated cellular components, or tissue slices. | -PIXE analysis of thin-film standards with biological samples quantifies elemental abundance to greater than 95% accuracy. By selecting X-rays of a specific energy, maps of element distributions can be obtained for the collected data set. Figure 13.13 illustrates this capability and shows the zinc distribution (attograms) in an 8 |lm by 8 |lm scan of a red blood cell.
STIM also utilizes a focused beam that is scanned across the sample. The energy loss is detected as a function of position as the accelerated ions (H+ or O3+) pass through the sample. This enables the mass of the sample to be accurately quantified without specific standards. We have demonstrated this on the analysis of isolated proteins with the quantification of 50 ng of bovine serum albumin. Figure 13.14 demonstrates the combination of PIXE to measure elemental quantities and STIM to measure protein mass. In Figure 13.14, we see the location of ferritin in a nitrocellulose membrane from a blot of an elec-trophoretic gel. The figure is oriented such that the gel's well is on the left. The top portion of the figure shows the location of several elements (phosphorus, sulfur, calcium, and iron) in the gel, while the bottom graph indicates the co-location of proteins and iron, relative to the starting well. The presence
of iron, as shown by the red line, is located at two positions; proteins are indicated by the blue line and are measured in ng (10-9gm). Ferritin, an iron-binding protein, is shown at ~0.8 cm from the well, the overlap of the protein and iron curves.
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