Imaging Technology: Aiding in Disease Detection

It is currently thought that around one-third of all proteins in the human body contain at least one metal ion. It is well-established that their biological activity is dependent on the presence of this metal. These ions can act as structural features or active sites for catalysis. Trace metals are so important to cell function that cell chemistry must be characterized not only by its characteristic genome and proteome, but also by the distribution of the metals and metalloids among different biomolecules - the "metallome."

Trace metals have long had an association with many neurological disorders, with most attention being directed to the ill effects of exogenous metals rather than essential elements. Parkinson's disease (PD) is a degenerative neurological disorder that is caused by the loss of dopaminergic cells within the pars compacta region of the substantia nigra.

Coincidental with the appearance of symptoms of the disease, an elevation of iron has been observed within the substantia nigra. Iron is suspected to be involved in the formation of reactive oxygen species within the substantia nigra, which is hypothesized to lead to the death of dopamine producing cells. It is unclear if the increased oxidative stress caused by Fe in PD is a cause or effect of the disorder.

Increased brain metal levels have also been associated with normal aging and Alzheimer's Disease (AD). Copper and iron levels both show marked increases with age and may adversely interact with the amyloid-beta peptide. This causes its aggregation and the production of neurotoxic hydrogen peroxide (H2O2), contributing to the pathogenesis of AD. Amyloid Precursor Protein (APP) possesses copper/zinc binding sites in its amino-terminal domain and in the amyloid beta domain.

Given that trace elements are critical for cell function and are associated with neurological disorders, it is desirable to gain knowledge about the former's distribution in soft tissue. Traditional staining methods have disadvantages, in that they are often not sensitive enough, or that the stains themselves introduce impurities. Although these methods are element specific, they are not capable of measuring multiple elements simultaneously, and often detect only certain species/oxidation states of the element of interest.

Inductively Coupled Plasma Mass Spectrometry
Mass Spectrometry (MS) methods are some of the most amenable to effective trace element detection in tissue. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has isotope specificity, versatility (virtually any element can be detected), high sensitivity, linear dynamic range (105 to 106) and non-dependence on the biological species of the trace element. This makes it an ideal tool for the detection of such elements in tissue samples.

ICP-MS has long been used for trace element analysis in environmental, semiconductor and geochemical applications. Samples are introduced into an argon plasma at a temperature range between 7500K -10,000K, resulting in decomposition and ionization (Figure 1).

The ions are extracted from the plasma through an interface (c) and focused by electrostatic lenses into a Collision/Reaction Cell (CRC). The CRC eliminates residual polyatomic ions that can interfere with masses of interest, ensuring that only elemental ions remain (d). From the CRC, the ions enter the mass filter (e) which separates them by mass to charge (m/z). This mass filter is most often a quadrupole, but magnetic sector and time-of-flight analyzers are also used in some ICP-MS. Ions leaving the quadrupole strike the electron multiplier (f ) that detects and amplifies the signal.

ICP-MS can be used as a detector for separation techniques such as liquid chromatography or capillary electrophoresis, and these hyphenated systems have been applied in recent years to the study of trace elements in biological samples. While such approaches have yielded valuable proteomics information, they are not amenable to accurately determining the spatial location of trace elements in situ in biological tissue. Current methods for this application include high-resolution x-ray spectroscopy such as the X-ray Absorption Near Edge Structure (XANES) technique, and X-Ray Fluorescence (XRF).

While these methods can detect as little as 0.1 pg and have a spatial resolution of five microns, a powerful synchrotron microbeam is required to generate X-rays of sufficient intensity to achieve this performance. As a result, the accurate spatial assessment of trace elements in tissue has been out of the reach of most laboratories until recently.

Elemental Bio-Imaging
An Elemental Bio-Imaging (EBI) system has been developed that interfaces a Laser Ablation (LA) station with the ICP-MS to provide virtual images of trace elements in tissue slices. A laser is rastered across a sample one line at a time, ablating tissue from the surface in 4 to 100 micron sections (Figure 2).

The ablated material is then swept into the ICP-MS, where the former's elemental composition is determined. In this way, an image can be built up by multiple raster lines, much in the same way that a dot matrix printer prints an image. The images are processed and displayed as color maps, with high concentrations usually designated in red and low concentrations in blue. Since the ICP-MS is a multi-element analyzer, it is possible to build maps for many elements simultaneously.

LA-ICP-MS is a more cost effective approach to trace element imaging in tissue than the current x-ray-based techniques. It eliminates much of the sample preparation associated with more traditional techniques of excising, grinding and extracting trace elements from the tissue before analysis. While this application of existing technologies is still in development, it holds the promise of providing rapid analysis times, with little operator input or expertise required.

Monitoring Disease Progression
This approach to the spatial imaging of trace elements has already been used to gain insight into the aetiology of some diseases. Elemental bio-imaging has been used to monitor the changes that occur in brain tissue during the induction of Parkinson's disease in a mouse model system. Parkinson's is caused by the loss of pigmented dopaminergic neurons in the substantia nigra, a portion of the midbrain, and is characterized by the reduced production of dopamine.

The cause of this cell death is still unknown, but there are suggestions that it may be due to an active toxic process involving oxidative stress that may be facilitated by an interaction between iron and dopamine in the Substantia Nigra (SN) region of the brain. The substantia nigra is normally rich in iron, and a further accumulation of iron has been observed in Parkinson's disease, as well as animal models of the disease.

Parkinson's disease can be induced in the rodent brain via the injection of the neurotoxin 6-hydroxy-dopamine (6-OHDA) into SN (10), in this case into one hemisphere only. Elemental bio-imaging of tissue slices after the injection reveals increased iron levels both at the injection site and in the substantia nigra, versus the un-lesioned hemisphere (Figure 3).

The accumulation of iron at the injection site could be due to blood entering the injection tract and the persistence of haemoproteins. Other trace metals such as manganese, copper and zinc do not show any significant changes in distribution after injection (data not shown). This accumulation of iron in the substantia nigra is of interest as a therapeutic target, as it may be the causal component of the death of substantia nigra neurons.

The potential utility of LA-ICP-MS as a tool for the detection of calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphates (BCP) in rheumatology has also been investigated. The concurrence of BCP (hydroxyapatite, octacalcium phosphate, tricalcium phosphate) and CPPD crystals and degenerative joint disease has been well established.

Although there is ample data to support the role of BCP crystals in cartilage degeneration, it is still unclear whether other calcium-containing crystals play a direct driving role in disease conception and progression, or are merely markers of joint damage.

Knee cartilage sections from a patient with osteoarthritis were obtained. The elemental distribution maps of calcium, phosphorus, magnesium and strontium are presented in Figure 4.

The corresponding regions of relatively high calcium and phosphorus intensities in the cartilage may be representative of calcium phosphate-based crystal deposits. A high frequency of CPPD crystals in articular tissue removed from osteoarthritis hips and knees has been reported in the literature. Other elements including copper, iron, and selenium did not follow the same trend in distribution.

While the virtual images of trace elements can reveal a wealth of information about changes in tissue, they must also be quantitative in order to be ultimately useful. A quantification method that is based on spin coated calibration standards has been developed.

Figure 5 illustrates the concept of ablation of the sample together with a film containing an internal standard. Quantitative data is produced by comparing the ratio of the trace elements in the tissue sample to that of the internal standard with the ratio obtained from spin coated slides with a known amount of trace element. This approach generates calibration curves that compensate for the differences in instrumental drift and the efficiency of transfer of the ablated material to the ICP-MS.

Trace elements play a key role in a wide range of biological processes. A full understanding of those processes requires knowledge of the genomics and proteomics of the organism, as well as the distribution and concentration of trace elements.

LA-ICP-MS may be utilized for the in situ analysis of trace metals in biological tissue. Using Elemental Bio-Imaging, isotope-specific maps of the spatial distribution of trace elements, particularly metals within thin tissue sections can be constructed. These images may be employed to probe the mechanism of many diseases in which metals are suspected to be involved such as Parkinson's disease and Alzheimer's disease.

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In collaboration with Agilent Technologies, the University of Technology Sydney (UTS) has established the Elemental Bio-imaging facility, a research effort utilizing laser ablation-ICP-MS (LA-ICP-MS) to study trace metals and other elements in tissue, and their effects on health.

UTS is providing the facility and scientific staff to perform the research to develop this application of ICPMS, and Agilent is providing the instrumentation, as well as funding for project work and postgraduate student support.

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