Chapter 5 – Detection and Imaging Tools that Use Nonoptical Waves  181

the transmission of x-​rays through the sample. However, this transmission mode has too low

a sensitivity for the often meager concentration of metals found in many biological materials,

and in this instance, x-​ray fluorescence emission is a better metric, with the detector pos­

ition at 90° from the incident beam. Detectors are typically based on doped semiconductor

designs such that the absorption of an x-​ray photon at a p–​i–​n junction of PIN diodes (where

i is an insulating layer between positive p and negative n doped regions) creates a hotspot of

electron–​hole pairs, which can be detected as a voltage pulse.

X-​ray photoelectron spectroscopy (XPS) is an alternative technique to XAS. A competing

mechanism to X-​fluorescence following absorption of an x-​ray photon by an atom is the

emission of a so-​called Auger electron—​the term Auger electron spectroscopy is synonymous

with XPS, and often the technique is abbreviated simply to electron spectroscopy. Here, low-​

energy x-​rays, either from an x-​ray tube or synchrotron source, are used to stimulate the

photoelectric effect in sample atoms, and these photoelectrons are detected directly by a

high-​resolution electron spectrometer, and electron intensity is determined as a function

of energy. The penetration distance of photoelectrons is ~10 nm in a sample, and so XPS

renders surface information from a sample, in addition to requiring high-​vacuum conditions

between the sample and detector. XPS is less sensitive than XAS with therefore more limited

application, but as a tool potentially offers advantages over XAS in being able to utilize x-​ray

tube sources as opposed to requiring access to a synchrotron facility. The temporal resolution

of XPS is in femtoseconds, which is ideal for probing electronic resonance effects in complex

biomolecules; for example, this has been applied to investigating different forms of chloro­

phyll (see Chapter 9), which is the key molecule that absorbs photons coupled to the gener­

ation of high-​energy electrons in the process of photosynthesis in plants and several other

unicellular organisms (see Chapter 2).

In principle, it offers a similar elemental signature, sensitive enough to detect and dis­

criminate between the energies of the photoelectric emissions from all atomic nuclei with

an atomic number Z of at least 3 (i.e., lithium and above). A limitation for probing biological

material is that the sample must be in a vacuum to minimize scatter of the emitted electrons;

however, it is possible to keep many samples in a cold, glassy, hydrated state just up the point

at which XPS is performed, before which ice sublimes off at the ultralow pressures used. XPS

has been applied to quantify the affinity and geometry of metal binding in protein complexes

and larger scale biological structures such as collagen fibers but is also used in elemental ana­

lysis on wood/​plant matter and teeth (e.g., in bioarcheology investigations).

5.3.6  RADIATION DAMAGE OF BIOLOGICAL SAMPLES BY X-​RAYS

AND WAYS ON HOW TO MINIMIZE IT

A significant limitation to the use of x-​ray photon probes in biological material is the high

likelihood of stochastic damage to the sample. X-​ray–​associated radiation damage is pri­

marily due to the photoelectric effect. As we have seen, the initial absorption event of an

x-​ray photon by an atom can result in the complete ejection of an inner shell electron. The

resulting atomic orbital vacancy is filled by an outer shell electron. For high-​atomic-​number

elements, including many metals, there is a significant likelihood of subsequent x-​ray fluor­

escence, however, for low Z elements, many of which are biologically, highly relevant such

as C, N, and O, but also S and P; the electron ejection energy is transmitted to an outer shell

electron, which is ejected as an Auger electron in a process, which takes ~10⁻¹⁴ s.

This photoelectric effect can then lead to secondary electron ionization in other nearby

atoms by electron-​impact ionization, resulting in the formation of chemically highly reactive

free radicals. It is these free radicals that cause significant damage through indiscriminate

binding to biological structures. Cooling a sample can minimize this damage simply by redu­

cing the rate of diffusion of a free radical in the sample, and it is common to cool protein

crystals in x-​ray crystallography with liquid nitrogen to facilitate longer data acquisition

periods.

Use of smaller crystals (e.g., down to a length scale of a few tenths of microns) also reduces

the effect of x-​ray radiation damage. This is because the loss of photoelectrons from a crystal