An MRI time course of 512 echo-planar images (EPI) in resting human brain obtained every 250 ms reveals fluctuations in signal intensity in each pixel that have a physiologic origin. Regions of the sensorimotor cortex that were activated secondary to hand movement were identified using functional MRI methodology (FMRI). Time courses of low frequency (< 0.1 Hz) fluctuations in resting brain were observed to have a high degree of temporal correlation (P < 10(-3)) within these regions and also with time courses in several other regions that can be associated with motor function. It is concluded that correlation of low frequency fluctuations, which may arise from fluctuations in blood oxygenation or flow, is a manifestation of functional connectivity of the brain.

Functional connectivity in the motor cortex of resting human brain using echo-planar MRI.

Approximately a third of the human proteome contains metal cations, either in form of cofactors with catalytic functions, or as structural support elements.1,2 To guarantee a proper maintenance of this metal ion pool, both at the cellular and whole organism levels, nature has evolved a highly sophisticated machinery comprised of a complex interplay between DNA, proteins, and biomolecules.3 Over the past decades, a steadily growing number of diseases have been identified, which are characterized by metal imbalance in cells and tissues. Among the most prominent examples rank Alzheimer’s disease and Parkinson’s disease, two neurodegenerative disorders that involve abnormal accumulation of transition metals in brain tissue.4 While some progress has been made at understanding the molecular basis of these disorders, many important questions remain unanswered. For example, little is known about the cellular structures that are involved in transiently storing metal ions prior to their incorporation into metalloproteins or the fate of metal ions upon protein degradation. An important first step towards unraveling the regulatory mechanisms involved in trace metal transport, storage, and distribution represents the identification and quantification of the metals, ideally in context of their native physiological environment in tissues, cells, or even at the level of individual organelles and subcellular compartments.

Since the inception of the first histochemical methods for the microscopic demonstration of transition metals in tissues more than 140 years ago,5 many highly sensitive microanalytical techniques and instruments have been developed for the in situ analysis of trace metals. The aim of this review is to provide an overview of the most recent achievements in trace metal imaging while at the same time also offering a historical perspective of this rapidly evolving research field. Although this survey has been structured according to the various analytical techniques, particular emphasis is given to the biological background for a better understanding of the context and importance of each discussed study.

An overview of the most important microanalytical techniques currently available for the in situ detection of trace metals in cells and tissues is compiled in Table 1. Depending on the task, each technique may offer specific advantages and, of course, also disadvantages. Currently, synchrotron- and focused ion-beam microprobes presumably offer the best combination of sensitivity and spatial resolution; however, the ionizing high-energy excitation beam is not compatible with studying live organisms. Conversely, techniques that have been specifically developed for physiological imaging in clinical medicine, notably magnetic resonance imaging and positron emission tomography, inherently offer only a low spatial resolution and are merely suitable for obtaining information at the organ or tissue level. Although fluorescence microscopy based methods provide very high sensitivity down to the single molecule level while being at the same time compatible with live cell and tissue studies, scattering and limited penetration depth renders these techniques unsuitable for imaging opaque specimens. There are also important differences regarding the type of quantitative information that can be gained by each of these analytical techniques. For example, the histochemical detection with chromogenic and fluorogenic dyes relies on a competitive exchange of the metal ion within its native environment, most likely coordinated to endogenous ligands. Depending on the exchange kinetics and thermodynamic affinity of the histochemical indicator, only a fraction of the total metal ion contents in a cell or tissue can be probed. Nevertheless, this kinetically labile pool is particularly of interest in context of understanding the uptake, distribution, and regulation of trace elements at the cellular level, and in this regard, these methods offer unique opportunities to dynamically image metal ion fluxes in live cells with high sensitivity and spatial resolution. At the same time, organelles and proteins of interest can be readily labeled with genetically encoded green fluorescent protein tags,12 thus providing direct insights into dynamic processes within a larger cellular and biochemical context. In contrast, similar correlative information is difficult to gain with the fully quantitative micro beam methods, which require xenobiotic elemental tags for identifying subcellular structures. Autoradiographic tracer experiments offer much improved resolution over PET; however, the technique is only applicable to fixed or frozen tissues and cells. Furthermore, tracer studies cannot provide direct information regarding the endogenous metal composition of cells or tissues, and are therefore primarily limited to metal uptake, distribution, and release studies. Finally, mass spectrometric analyses are surface-based methods that destroy the sample while measuring its elemental composition. Clearly, only the combination of several analytical techniques and specific biochemical studies may lead to a fully comprehensive analysis of a biological system.



Table 1

Spatially resolved microanalytical techniques for in situ imaging of trace metals in biology.6–11




2. Histochemical Techniques
Histology is the branch of biology dealing with the study of microscopic anatomy of cells and tissues of plants and animals. Histological studies are typically carried out on thin sections of tissue or with cultured cells. To visualize and identify particular structures, a broad spectrum of histological stains and indicators are available. Among the most widely used dyes are hematoxylin and eosin, which stain nuclei blue and the cytoplasm pink, respectively.13 The history of detecting biological trace metal by histological methods dates back more than 140 years. Although these techniques have been today mostly replaced by the much more sensitive modern analytical methods described in this review article, histochemical approaches for visualizing metals mark the very beginning in the exploration of the inorganic physiology of transition metals. Given this special place in history, we deemed it necessary to briefly review some of the early achievements in this field.

2.1. Chromogenic Detection with Chelators and Ligands
Ever since the inception of Perls Prussian blue method for staining of non-heme iron, numerous indicators have been developed for the in situ visualization of trace metals in biological tissues and cells.13 Due to their limited sensitivity; however, most of these techniques were only suitable for the diagnosis of pathological conditions, typically associated with excess metal accumulations, thus preventing their application for routine staining of normal tissue. Furthermore, because the dyes are engaged in a competitive exchange equilibrium with endogenous ligands, histological stains are not suitable for the analytical determination of the total metal contents in tissues and thus limited to the visualization of the histologically reactive fraction of loosely bound labile metal ions.



2.1.1. Histochemistry of Iron 
The histochemical demonstration of labile iron reported by Perls in 1867 is among the earliest accounts describing the in situ visualization of a trace metal in biological tissues.5 The method was originally described by Grohe, who observed the formation of a blue coloration when he treated cadaver tissues with potassium ferrocyanide in acidic solution.14 Due to its low cost and simplicity, the technique is still used today for the histological visualization of non-heme iron. Some variations focused on optimizing the concentrations and proportions of the reagents,15–17 among which Lison’s protocol17 appears to be most popular today. An intensification of Perls’ staining can be obtained by exploiting the use of ferric ferrocyanide in catalyzing the oxidation of diaminobenzidine (DAB) to polymeric benzidine black by hydrogen peroxide.18

An alternative method employs the reaction of ferricyanide with Fe(II) resulting in Turnbull blue.19 Since almost all of the Fe in tissues is in the ferric form, the staining procedure requires the in situ conversion of Fe(III) to Fe(II) with ammonium sulfide.15 Due to often incomplete reduction, the method never gained much attention. More recently, an application of Turnbull blue, named the ‘perfusion Turnbull method’ has been developed, where in vivo perfusion of acidic ferricyanide is followed by DAB intensification.20 The direct in vivo perfusion avoids artifacts associated with tissue fixation, including the loss of loosely bound iron and oxidation of Fe(II) to Fe(III). Similarly, Perls method was modified by employing in vivo perfusion with acidic ferrocyanide. Both methods are capable of identifying organs and tissues containing histochemically reactive iron over a broad pH range, including the low endosomal pH.21,22

The history of iron histochemistry would be incomplete without mentioning Quincke’s method, which employed ammonium sulfide for the precipitation of tissue iron as its sulfide.23 A detailed account on the various techniques, including a comprehensive historical overview of non-heme iron chemistry, has been recently published.24

In Situ Imaging of Metals in Cells and Tissues

Ion beam induced surface and interface engineering

Effects of thirty elements on bone metabolism

This article presents an overview of the recent developments and requirements in radiotherapy dosimetry, with particular emphasis on the development of optical fibre dosemeters for radiotherapy applications, focusing particularly on in vivo applications. Optical fibres offer considerable advantages over conventional techniques for radiotherapy dosimetry, owing to their small size, immunity to electromagnetic interferences, and suitability for remote monitoring and multiplexing. The small dimensions of optical fibre-based dosemeters, together with being lightweight and flexible, mean that they are minimally invasive and thus particularly suited to in vivo dosimetry. This means that the sensor can be placed directly inside a patient, for example, for brachytherapy treatments, the optical fibres could be placed in the tumour itself or into nearby critical tissues requiring monitoring, via the same applicators or needles used for the treatment delivery thereby providing real-time dosimetric information. The article outlines the principal sensor design systems along with some of the main strengths and weaknesses associated with the development of these techniques. The successful demonstration of these sensors in a range of different clinical environments is also presented.

/pdf/a-review-of-recent-advances-in-optical-fibre-sensors-for-in-1nnarnpzbz.pdf

A review of recent advances in optical fibre sensors for in vivo dosimetry during radiotherapy

Metal deposition at the bone-cartilage interface in articular cartilage.

A comparative study of the structural changes induced in Al/Ti and AlN/TiN multilayers by argon ion irradiation is presented. The layers were deposited on Si substrates, by ion sputtering in case of pure metals, and reactive ion sputtering in case of metal-nitrides, to a total thickness of ∼270 nm. The multilayered structures consisted of 10 alternative ∼27 nm layers of each component. For ion irradiation, we used 200 keV Ar + ions having a projected range around mid-depth of the multilayered structures. Implantations were performed at room temperature, to the fluences from 5 × 10 15 to 4 × 10 16  ions/cm 2 . Structural characterization included Rutherford backscattering analysis and transmission electron microscopy. The obtained results demonstrate that AlN/TiN multilayered system exhibits a much higher irradiation stability compared to the Al/Ti system. In Al/Ti multilayers, we observe a progressed intermixing with increasing the ion fluence, the behavior being closer to the ballistic than to the thermal spike model. In AlN/TiN system, no interface mixing was registered for any of the applied irradiation fluencies. Different behavior compared to Al/Ti system is assigned to immiscibility of AlN and TiN.

On the ion irradiation stability of Al/Ti versus AlN/TiN multilayers

The influence of ion species on the ion-cut, or smart-cut© process in GaAs has been investigated in this paper. Semi-insulating GaAs, implanted with H2+, He+ and Ne+, were studied by Rutherford backscattering spectroscopy (RBS) in channelling mode, to investigate the lattice disorder produced by implanting different ion species. The RBS spectra show that surface dechannelling is enhanced where blisters are present. The samples were also studied by Nomarski microscopy and atomic force microscopy to investigate the effect of the ion species on the blistering process. It was found that the ion-cut process can be achieved at lower doses by using the implantation of He alone rather than H. Our results indicate that the implantation of Ne is not a suitable alternative to H or He implantation.

Characterising ion-cut in GaAs by Rutherford backscattering spectroscopy

Proceedings of the First International Conference on Biomedical Applications of High Energy Ion Beams.

The dependence of hydrogen flux and substrate temperature during implantation on the smart-cutprocess in GaAs have been investigated in this paper. The lattice disorder in the samples was studied by ion beam analysis. It was found that both the flux and the implant temperature are significant for the smart-cut process. As the flux increases, the degree of exfoliation and the mean blister size increases, while the number density of blisters falls, indicating that blister evo- lution is enhanced at high dose rates.

The influence of the ion implantation temperature and the dose rate on smart-cut© in GaAs

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