**3.3.2 Viewing copper differently – Dramatic fluxes of copper**

It was reported in 2006 by Gitlin et al. that copper in hippocampal neurons appeared to be exported following NMDA-receptor stimulation (Schlief, Craig et al. 2005). The use of Cu-64 to try to measure this export made it somewhat difficult to determine exactly how much of the cellular copper was exported, or image exactly where the copper was. But, clearly, new roles for copper were emerging.

Shortly after this, Finney et al. reported that a dramatic efflux of copper occurs during the angiogenic process of tubulogenesis, or the process by which new capillaries are formed. As mentioned earlier, copper had long been known to be important to angiogenesis, and thus also to the growth of cancerous tumors that rely upon a growing blood supply. By directly imaging the tubulogenesis process, at fixed points, using X-ray fluorescence microscopy scientists found that between 80-90% of the cell's copper was exported at early points, and then taken back up later in the growth of capillary-like structures (Finney, Mandava et al. 2007). Exactly why this happens remains a mystery, and has sparked new efforts in the development of tools for metalloproteomics (Finney, Chishti et al. 2010). From this, one might speculate that a role for copper in intercellular signaling, of some sort, may exist.

Taking this technique, and applying it back to the same sort of systems which Gitlin et al. had examined, leads to another remarkable finding. As shown in Figure 2, a typical SH-sy5y cell, the majority of cellular copper is typically localized in the perinuclear area in neuronal cells. Upon stimulation, the copper may be seen to relocalize such that a significant increase in the fraction of cellular copper that is along the dendrites of the cell is seen. Not only can fluxes of copper be seen in hippocampal neurons, but they are dependent on calcium, and induced by extracellular stimulation (Dodani, Domaille et al. 2011). Clearly, undiscovered roles for copper as an important part of cell signaling exist. And X-ray fluorescence imaging is enabling our further understanding of them. As roles for metals such as this are further defined, they hold the potential to reveal patterns and signatures that may become the biomarkers of the future.

Inorganic Signatures of Physiology: The X-Ray Fluorescence Microscopy Revolution 85

cells at the scale of 10's of nanometers, where we can begin to see within the mitochondrion, for example, will reveal many new and exciting things about the cell biology of metals.

Another important consideration in the context of biomarkers is speed. X-ray fluorescence imaging is currently quite slow. Samples are raster-scanned through an X-ray beam with dwell times of 1s or more per pixel. With potentially hours of scan time required for imaging a single cell, good statistical sampling is difficult to achieve. What is needed for this? Some current areas of development may help, particularly the development of fast fly-scanning data acquisition, where samples are raster scanned continuously and fluorescence information is recorded 'on-the-fly'. Another area with promise for speed is the development of microfluidic devices that will enable X-ray fluorescence spectra of whole cells to be individually captured while flowing in a stream, thus allowing measurement of at least the total metals in individual cells over populations of hundreds of cells. As techniques like these emerge, the promise of X-

This work was supported by the DOE Office of Science under contract DE-AC02- 06CH11357. The author thanks all of her collaborators, especially those cited here, for

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**4.3 Need for other data acquisition schemes to improve statistics** 

ray fluorescence imaging for diagnostics comes closer to a reality.

**5. Acknowledgment**

**6. References** 

sharing the excitement of their research.

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Fig. 2. Copper localizes peripherally to the nucleus in a cell
