**1. Introduction**

76 Biomarker

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#### **1.1 Metals as an essential, yet potentially toxic part of cellular chemistry**

Metals are vital to nearly all the life processes within the cell. It is estimated that nearly a third of cellular proteins bind metals. Yet, the very same properties of these metal ions which make them so useful, also makes them potentially hazardous within the cell. Endogenous metals, such as copper, zinc, and iron are also potentially toxic, performing deleterious redox chemistry if not carefully controlled and regulated(Finney and O'Halloran 2003). The intricate cellular machinery that manages these metals are collectively known as the metal homeostasis and trafficking proteins of the cell. While we have learned much with respect to how the cell partitions and allocates metals, and at times attempted to define the 'concentration' in various compartments of the cell, instead we have begun to see how illdefined a 'resting' condition really is, and how much the partitioning of cellular metals can change (Dodani, Leary et al. 2011; Qin, Dittmer et al. 2011).

What is it about metals which make them so critical, and so useful? Unlike other elements, biological metals, and particularly the first row of transition metals in the periodic table, have a partially filled d-shell of orbitals. This gives them multiple semi-stable oxidation states under ambient conditions. And the changes between these states provide reduction and oxidation potential for the chemistry of the cell (Bertini, Gray et al. 2007). For example, iron, as part of hemoglobin, shuffles between a (II) and (III) oxidation state to perform the vital act of delivering oxygen throughout the body. However, the same element, iron, if it were a 'free' aquo- ion in the cytoplasm of the cell would likely undergo Fenton chemistry, and these same changes in oxidation state would give rise to the generation of radicals that would damage the cell.

Still, biology manages, most of the time, to work. The efficient chemistry of these life processes often exceeds our own ability to accomplish their work synthetically. Unraveling the ways in which the chemistry of these metals is controlled promises not only to improve what we know about chemistry, accomplishing important reactions such as the oxidation of methane or the fixation of nitrogen in new and more efficient ways, but also to remarkably improve our understanding of biology, and our ability to manipulate it as well.

technique.

makes such surprising findings possible.

**1.4 Are these signatures diagnostic?** 

physiological state or disease condition.

**2. Overview of X-ray fluorescence imaging** 

**2.1 Prinicples of biological X-ray fluorescence imaging** 

metals, they excite the electrons bound to the atom directly.

**2.1.1 Basics of X-ray fluorescence** 

Inorganic Signatures of Physiology: The X-Ray Fluorescence Microscopy Revolution 79

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

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

According to the National Cancer Institute, a biomarker is "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease. A biomarker may be used to see how well the body responds to a treatment for a disease or condition. Also called molecular marker and signature molecule". In this sense, a biomarker is something that is diagnostic – serving as an indicator of a

The cellular distributions of metals clearly have the potential to fulfill this role. As we learn more about the distributions and compartmentalization of cellular metals, and how these

Fluorescence exists in many familiar forms. It is the absorption of light at one wavelength, and its re-emission as light of a longer wavelength (with less energy). It can be seen when you put certain laundry detergents under a simple black-light, which you cannot see with your eyes, and watch optical light come back out, making it 'glow'. Optical fluorescence like this is also a critical tool in almost all of biology, where even the most complex optical fluorescence microscopes still use monochromatic light and emission filters to image optically-fluorescent dyes and protein labels, revealing information about cellular structures. X-ray fluorescence imaging is fundamentally no different from this. However, unlike optical light which excites vibrational states, X-rays are of such energy that, for

The energy and wavelength of light are inversely related, as follows from Equation 1, where

E is energy, h is Planck's constant, c is the speed of light, and is the wavelength.

change in various conditions, it is increasingly clear that signatures should exist.
