**1.2 The location of metals, and bioavailability, within the cell regulates cell function**

Metals serve roles in thousands of proteins and enzymes, and are found in various species throughout the mammalian cell. Good recent reviews of biological copper (Boal and Rosenzweig 2009; Banci, Bertini et al. 2010; Lutsenko 2010), iron (Kosman 2010), and zinc (Eide 2006; Tomat and Lippard 2010) exist. Our understanding of the location and bioavailability of metals, until rather recently, was mainly accessible by studying the properties of the proteins that bind them.

For example, one role of copper is as an important component of cytochrome c oxidase, which performs respiration in mitochondrion (Tsukihara, Aoyama et al. 1995; Tsukihara, Aoyama et al. 1996). From this as well as other examples, we know copper to be an important component in the mitochondrion. Among its many roles, zinc is a critical structural component of zinc-finger proteins, which regulate transcription in the nucleus. A significant reference of zinc-binding proteins is available for understanding the compartments of the cell where zinc may be found (Vallee and Auld 1990). Taken together, this kind of indirect knowledge leads to a coordinated, systems-based approach to understanding the location of cellular metals, as has been recently applied in the case of copper (Banci, Bertini et al. 2010).

These indirect approaches are also useful in understanding bioavailability of metals. For example, based upon the measurement of the metal-binding constant of superoxide dismutase we have been able to extrapolate that there is no free copper in the cytoplasm of yeast (Rae, Schmidt et al. 1999). Additionally, measurement of the zinc-binding potential of the CueR copper regulatory protein demonstrated that there was no 'free' copper in the cytoplasm of E. *coli* bacteria (Changela, Chen et al. 2003), setting the window of such ions at less than one atom per cell.

In addition to achieving the chemistry vital to the cell, metalloproteins open up a new avenue of cellular regulation. Not only can their activity be up- or down-regulated by changes in the expression level, but also by changes in the availability of their metal cofactors. An elegant illustration of this is the work of Tottey et al., which found, in examining the periplasm of the bacteria *Synechocystis* PCC 6803 that compartmentalization can be used to keep competitive metals out of the 'wrong' nascent proteins (Tottey, Waldron et al. 2008). In another, more clinical example, it has long been known that copper availability can modulate the growth of tumors in the body (Pan, Kleer et al. 2002). When copper is depleted by adminstering copper-chelating compounds such as tetrathiomolybdate to the patient, the growth of new blood vessels as well as the tumors that rely on them is inhibited.

#### **1.3 We can now visualize changes in cellular metal distributions, and their signature patterns, during physiological changes**

Recent technological advances have also made it possible to directly visualize metals within cells at the sub-cellular level. As early as the 1980's, advances in microanalysis were enabling the development of electron microscopy capable of compositional analysis – or the ability to distinguish the chemical composition of samples at the cellular or subcellular level. An excellent example of this is the work of Peter Ingram and Ann Le Furgey (Ingram, Shelburne et al. 1999). One of the limitations of electron microscopy is the thickness of samples, which generally must be no more than 100 nm thick, requiring specialized sample preparation.

On the other hand, hard X-ray microscopy (~10 keV or greater incident energy), generally does not face this limitation. The focal depth is on the order of 200 – 300 microns, and anything thinner than this will simply appear as 2-D projection of the volume. Thus, as zone plate technology and third generation synchrotron sources have developed, the minimal sample preparation required has made X-ray fluorescence microscopy a very accessible technique.

The accessibility of X-ray fluorescence microscopy, with relatively simple sample preparation and publically available synchrotron facilities, has facilitated its application – even to areas quite clinical and far removed from the physics of the synchrotron facilities that support it. The findings, some of which are highlighted below, have at times been startling. Could 80% of the cell's copper simply be exported during the angiogenic process of tubulogenesis (Finney, Mandava et al. 2007)? Isn't this a drastic commitment of energy? Despite clinical findings regarding chelation, as mentioned earlier, it is doubtful any scientist would have predicted this. But, it is precisely the directness of these methods which makes such surprising findings possible.
