**3.3.1 Established, enzymatic roles for copper**

82 Biomarker

were the ones to differentiate first. This exciting finding brings us to a new understanding of how little we know about the majority of nuclear zinc, as well as the roles of metals during

Fig. 1. Correlating XRF and immuno-fluorescence images of human embryonic stem cells.

At the same time, zinc plays many roles in the cell, as well as roles even outside of the cell. An elegant example of work utilizing X-ray fluorescence to better understand zinc physiology is reported by the Kelleher group in their studies of lactation (McCormick, Velasquez et al. 2010). In contrast to the coordination of zinc throughout much of the cell, zinc in milk is significantly associated with lower molecular weight molecules – at relatively high concentration relative to other essential metals. And it is not known how the mammary gland regulates the transfer of zinc into milk. By directly imaging the zinc in mammary tissue of both lactating and non-lactating mice, the researchers were able to demonstrate that zinc associates with a distinct peri-nuclear pool. Further, through experiments utilizing the chemical indictor Fluo-zin-3, which is a zinc indicator, together with dyes for the endoplasmic reticulum, mitochondrion, and Golgi, optical fluorescence microscopy indicated that this zinc was 'labile' on account with its ability to bind the zinc dye, and at least partially associated with the Golgi complex. This work represents some of the first Xray fluorescence imaging of mammary tissue at the sub-micron scale, and suggests that the pathways for zinc export during lactation likely are similar to those utilized in the prostate. Another particularly surprising finding from direct X-ray fluorescence imaging of metals in cells relates to fertilization. Work by a team of scientists from Northwestern University has recently shown that the accumulation of zinc is essential for fertilization (Kim, Vogt et al. 2010). In this work, single-cell elemental analysis of mouse oocytes by X-ray fluorescence microscopy revealed a 50% increase in total zinc content within the 12-14-h period of meiotic

differentiation.

Copper is widely used in biology for enzymatic chemistry. Its ability to cycle between (I) and (II) oxidation states makes it particularly useful for reduction and oxidation chemistry. It is used to activate oxygen, detoxify radicals, and in mitochondrial function. Yet, for as much as is known about copper, direct X-ray fluorescence imaging is revealing new roles, and changes in distributions that may have the potential to serve as biomarkers of the future.
