**2.1.1 Basics of X-ray fluorescence**

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.

Inorganic Signatures of Physiology: The X-Ray Fluorescence Microscopy Revolution 81

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

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

Zinc has long been known to play an important role in biology. Studies of the biochemistry of zinc may well have first begun in the 1950's, with the study of metallothionein. We now know that zinc plays both structural roles, such as in zinc finger proteins, and catalytic roles such as it does in carbonic anhydrase. Yet, there remains much to learn about this metal. Results from direct X-ray fluorescence imaging of this element in cells may indeed have

One of the first findings regarding zinc (utilizing sub-micron X-ray fluorescence imaging) was that it may be involved in cell differentiation, particularly looking at HL-60 cells (Glesne, Vogt et al. 2006). In examining the growth of human embryonic stem cells, taking a systems biology approach to examining entire colonies of cells and all the first row transition metals, we also found that the amount of zinc present in cells directly correlated with their differentiation (Wolford, Chishti et al. 2010). The images in Figure 1, of stem cells differentiated with retinoic acid, are particularly illustrative. Loss of Oct4 (pink) is associated with higher zinc (red in 'Zn' panel). This was found to be true regardless of the method of differentiation, or whether the cells at the outer edge or at the center of the colony

selenium at the subcellular level for better understanding of mammalian biology.

and sensitivity, will lead to a greater understanding of selenium biology.

**3.2 Case 2: A role for zinc in cell fertilization, differentiation, lactation** 

**3.2.1 A historical perspective on the cell biology of zinc** 

revealed that there is still much to learn.

**3.2.2 A new view of zinc from XFM** 

**3.1.2 What XFM has revealed** 

$$\mathbf{E} = \hbar \times \mathbf{c} \;/\; \lambda \tag{1}$$

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.
