**3.1.2 What XFM has revealed**

80 Biomarker

Looking at this equation, it is simple to see that if the energy of an incoming photon is decreased, such as might happen when it runs into an electron, the photon is not only reduced in energy but its wavelength will increase, changing its 'color'. In fact, the photon may excite the electron into a higher energy state. When this happens, it leaves behind an opening, or 'hole' in the electron shell. Since the spacing of orbitals, or the difference in energy between them, is constant for a particular metal atom, when an electron 'falls' back down in energy, into the 'hole' that was left behind, it emits light at a very characteristic energy – much like a pipe of a particular length on an organ plays a very specific note. It is this property of X-ray fluorescence, the fact that each element – zinc, copper, iron – will emit fluorescence at characteristic energies with specific relative intensities that are intrinsic to the metal itself, that makes it possible to distinguish the emission spectrum of iron from that of copper, for example. Or to distinguish how much of either one is present in a mixture.

Critical to the success of biological X-ray fluorescence imaging is preparation of samples which are both structurally and compositionally intact. At the same time, the samples must be preserved such that they can withstand the damaging potential of a focused X-ray beam. The most ideal way of ensuring this is to prepare samples which are frozen in a vitreous glass of ice. This avoids creating crystalline ice, which would break cellular structures and membranes and is not a simple task. Also, to keep the sample completely frozen without any recrystallization of the ice is not straight-forward. Current research is attempting to

Yet, much interesting and useful research has been done on dry samples. Recent studies examine either flash-frozen and freeze-dried samples, or chemically fixed and dried samples

Another consideration in examining biological samples is that the emission peaks of most common biological metals overlap quite strongly in the emission spectrum generated by energy-dispersive detectors. Thus, proper fitting of the data, including de-convolution of these peaks, is critical to correct assignment of intensity to a metal of interest. The development of software, particularly MAPS, has been of paramount importance in this field (Vogt 2003). Likewise, the selection and use of reference standards, to convert emitted

Selenium exists, in mammals, primarily as part of selenomethionine or selenocystine, and less abundantly as selenite, selenide, monomethylselenol, dimethylselenide, trimethylselenonium, L-selenomethionine (SM), Se-methyl-L-selenocysteine. Because of its chemical similarity, it is utilized by the body in many of the same pathways as sulphur. Some controversy has surrounded its use as a nutritional supplement in the prevention of

cancer – where it has been purported to function in an anti-oxidative capacity.

intensity to a calculated quantity, is also critical to proper analysis.

**3. The revolution: Peering into the unseen** 

**3.1 Case 1: The biology of selenium** 

**3.1.1 Overview of selenium biochemistry** 

**2.1.2 Special considerations for biological samples** 

achieve both of these.

(McRae, Bagchi et al. 2009).

E = *h* ×c / λ (1)

X-ray fluorescence has shed light on the biological roles of selenium in biochemistry. Kehr et al. obtained beautiful images, the first of their kind, of the selenium in sperm (Kehr, Malinouski et al. 2009). It had long been known that selenium is essential for sperm production and therefore fertility in mammals (Maiorino, Roveri et al. 2006). By directly imaging the selenium in the sperm at various stages of development, scientists found that a high and specific accumulation of selenium occurs during spermatid development. Further, they determined that it related to an increased need for the plasma selenoprotein SelP in order to produce additional mGPx4 protein. This work not only expanded the current understanding of selenium biology, but also demonstrated the utility of direct imaging of selenium at the subcellular level for better understanding of mammalian biology.

In another example, the effects of GPx1 deficiency were explored in mice. GPx1 is the major mammalian selenoprotein and it is expressed at a particularly high level in the liver (Malinouski, Kehr et al. 2011). The uniform distribution of Se in hepatocytes is consistent with the concept that XFM largely detects GPx1. In this work, it was found that in addition to homogenous signal from GPx1, the kidney also showed highly localized circular structures of Se surrounding proximal tubules. It was reported that this signal represents GPx3, which was secreted from these tubules and remained bound to the basement membrane. It represented approximately 20% of the Se pool in mouse kidney, and an even higher fraction in the kidney of the naked mole rat. This observation supports the postulate that the production of these two proteins, and their sources of selenium, are separate. The authors also postulate that advances in X-ray fluorescence imaging, increasing its resolution and sensitivity, will lead to a greater understanding of selenium biology.
