Today, the price of building a factory to produce submicron size electronic devices on 300 mm Si wafers is over billions of dollars. In processing a 300 mm Si wafer, over half of the production cost comes from fabricating the very-large-scale-integration of the interconnect metallization. The most serious and persistent reliability problem in interconnect metallization is electromigration. In the past 40 years, the microelectronic industry has used Al as the on-chip conductor. Due to miniaturization, however, a better conductor is needed in terms of resistance–capacitance delay, electromigration resistance, and cost of production. The industry has turned to Cu as the on-chip conductor, so the question of electromigration in Cu metallization must be examined. On the basis of what we have learned from the use of Al in devices, we review here what is current with respect to electromigration in Cu. In addition, the system of interconnects on an advanced device includes flip chip solder joints, which now tend ...

Recent advances on electromigration in very-large-scale-integration of interconnects

Recent years have seen significant progress in the field of soft- and hard-X-ray microscopy, both technically, through developments in source, optics and imaging methodologies, and also scientifically, through a wide range of applications While an ever-growing community is pursuing the extensive applications of today's available X-ray tools, other groups are investigating improvements in techniques, including new optics, higher spatial resolutions, brighter compact sources and shorter-duration X-ray pulses This Review covers recent work in the development of direct image-forming X-ray microscopy techniques and the relevant applications, including three-dimensional biological tomography, dynamical processes in magnetic nanostructures, chemical speciation studies, industrial applications related to solar cells and batteries, and studies of archaeological materials and historical works of art

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Nanoscale X-ray imaging

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

Rechargeable battery technologies have ignited major breakthroughs in contemporary society, including but not limited to revolutions in transportation, electronics, and grid energy storage The remarkable development of rechargeable batteries is largely attributed to in-depth efforts to improve battery electrode and electrolyte materials There are, however, still intimidating challenges of lower cost, longer cycle and calendar life, higher energy density, and better safety for large scale energy storage and vehicular applications Further progress with rechargeable batteries may require new chemistries (lithium ion batteries and beyond) and better understanding of materials electrochemistry in the various battery technologies In the past decade, advancement of battery materials has been complemented by new analytical techniques that are capable of probing battery chemistries at various length and time scales Synchrotron X-ray techniques stand out as one of the most effective methods that allow for near

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Synchrotron X-ray Analytical Techniques for Studying Materials Electrochemistry in Rechargeable Batteries

Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of deltaG++ = 44 kJ.mol(-1) and k(obs) approximately 10(5) s(-1) at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.

https://www.pnas.org/content/pnas/102/32/11179.full.pdf

Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy

The uptake of carcinogenic and mutagenic Cr compounds and the intracellular distribution of their biotransformation products in V79 Chinese hamster lung cells were studied by synchrotron-radiation-induced X-ray emission (SRIXE). SRIXE analysis was performed on whole cells that had been treated with either Cr(III) or Cr(V) 1,10-phenanthroline complexes, or Cr(VI). The high spatial resolution (0.3 µm) and elemental sensitivity (~10–15 g Cr/cell) of the technique provided detailed maps of Cr and other cellular elements in thin sections prepared from Cr(VI)-treated cells. The Cr carcinogen concentrated in P-rich regions corresponding to the nucleus, as well as other areas of the cell that are likely to correspond to organelles. This is the first study that has enabled the determination of the localization of the biotransformation products of Cr(VI) carcinogens in a target lung cell. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0343-5.

Hard X-ray microprobe studies of chromium(VI)-treated V79 Chinese hamster lung cells: intracellular mapping of the biotransformation products of a chromium carcinogen.

Synchrotron-based x-ray microbeam techniques have been used to map crystallographic strain and multilayer thickness in micro-optoelectronic devices produced with the selective area growth technique. Our main results show that growth enhancements in InGaAsP multilayer device material are different for well and barrier material. Comparison with a vapor-phase model for selective area growth suggests that this difference is due to different vapor-phase incorporation rates for the group III metals.

Synchrotron x-ray microdiffraction diagnostics of multilayer optoelectronic devices

We use Fresnel zone plates as focusing optics in hard x-ray microprobes at energies typically between 6 and 30 keV While a spatial resolution close to 01 {micro}m can currently be achieved, highest spatial resolution is obtained only at reduced diffraction efficiency due to manufacturing limitations with respect to the aspect ratios of zone plates To increase the effective thickness of zone plates, we are stacking several identical zone plates on-axis in close proximity If the zone plates are aligned laterally to within better than an outermost zone width and longitudinally within the optical near-field, they form a single optical element of larger effective thickness and improved efficiency and reduced background from undiffracted radiation This allows us both to use zone plates of moderate outermost zone width at energies of 30 keV and above, as well as to increase the efficiency of zone plates with small outermost zone widths particularly for the energy range of 6-15 keV

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Near-field stacking of zone plates in the x-ray range.

A hard x-ray scanning microprobe based on zone plate optics and undulator radiation, in the energy region from 6 to 20 keV, has reached a focal spot size (FWHM) of 0.15 μm(v)×0.6 μm(h), and a photon flux of 4×109 photons/sec/0.01%BW. Using a slit 44 meters upstream to create a virtual source, a circular beam spot of 0.15 μm in diameter can be obtained with a photon flux of one order of magnitude less. During fluorescence mapping of trace elements in a single human ovarian cell, the microprobe exhibited an imaging sensitivity for Pt (Lα line) of 80 attograms/μm2 for a count rate of 10 counts per second. The x-ray microprobe has been used to map crystallographic strain and multiquantum well thickness in micro-optoelectronic devices produced with the selective area growth technique.

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A hard x-ray scanning microprobe for fluorescence imaging and microdiffraction at the advanced photon source

A hard x-ray scanning microprobe based on zone plate optics and undulator radiation, in the energy region from 6 to 20 keV, has reached a focal spot size (FWHM) of 0.15 {micro}m (v) x 0.6 {micro}m (h), and a photon flux of 4 x 10{sup 9} photons/sec/0.01%BW. Using a slit 44 meters upstream to create a virtual source, a circular beam spot of 0.15 {micro}m in diameter can be obtained with a photon flux of one order of magnitude less. During fluorescence mapping of trace elements in a single human ovarian cell, the microprobe exhibited an imaging sensitivity for Pt (L{sub a} line) of 80 attograms/{micro}m{sup 2} for a count rate of 10 counts per second. The x-ray microprobe has been used to map crystallographic strain and multiquantum well thickness in micro-optoelectronic devices produced with the selective area growth technique.

/pdf/a-hard-x-ray-scanning-microprobe-for-fluorescence-imaging-1bpmmxwphj.pdf

W. Rodrigues

Papers

Hard X-ray microprobe studies of chromium(VI)-treated V79 Chinese hamster lung cells: intracellular mapping of the biotransformation products of a chromium carcinogen.

Synchrotron x-ray microdiffraction diagnostics of multilayer optoelectronic devices

Near-field stacking of zone plates in the x-ray range.

A hard x-ray scanning microprobe for fluorescence imaging and microdiffraction at the advanced photon source

A hard x-ray scanning microprobe for fluorescence imaging and microdiffraction at the Advanced Photon Source