2.1. Ex-Vivo Iron Detection and Measurement

We first discuss qualitative and quantitative ex vivo methods for studying tissue iron, which can be considered as the gold standard for studying iron chemistry and quantity.

Fe³⁺ staining (Perls’ Prussian blue) and Fe²⁺ staining (Turnbull’s blue) have been used for qualitatively exploring the distribution of iron in brain tissue. Because most iron in the brain is present as ferric (Fe³⁺) iron, Perls’ staining is the most commonly used technique to visualize iron histologically. In addition, immunohistochemical labeling is used for iron-related proteins, including ferritin [10,47,48], transferrin [47,49,50] and ceruloplasmin [51]. However, presence of ferritin does not necessarily reflect presence of iron, as has been shown in myeloid cells where ferritin can be present in the absence of iron [9].

A few spectrometric techniques are available to quantify iron in biological tissue, which are either based on the characteristic atomic mass [52] or element-specific characteristic X-rays emitted from atomic inner shell transitions [53]. Spectrometric methods measure iron irrespective of its chemical state, i.e., Fe²⁺ or Fe³⁺, thus quantifying the total iron content. Here is a brief outline of commonly used iron quantification methods:

Colorimetry: This iron-detecting approach requires a standard of the measured absorbance for known concentrations, thus making it problematic for comparing tissue samples of different measurements [47,54,55].

Atomic Absorption Spectrometry or Spectrophotometry (AAS): The technique equally requires standards with known analyte content to establish a relationship between measured absorbance and analyte concentration [56,57,58].

Instrumental Neutron Activation Analysis (INAA): The intensity and wavelength of the signal is element-specific and can be used to characterize the examined sample [59,60,61].

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [4,62,63]: ICP-MS has become the most prominent method to quantitatively correlate iron concentration and MR-signal, in particular iron and R2* [17,64,65,66,67], although a quantitative mapping of the elemental distribution is not possible. However, ICP-MS in combination with laser ablation (LA-ICP-MS) allows for microlocal element analysis [68,69]. LA-ICP-MS has been use to quantitatively map the iron distribution of brain tissue [41,70].

X-ray Fluorescence Spectrometry (XRF) or Rapid Scan X-ray fluorescence spectrometry (RS-XRF) [71,72]: This technique allows for iron mapping and experiments have been conducted at two different locations; at the Stanford Synchrotron radiation laboratories (SSRL) [73] or more recently at the new Diamond light source in Oxfordshire, UK [74].

Proton-Induced X-ray Emission (PIXE) [75,76,77]: A similar principle as XRF allowing to create iron maps of brain tissue [78,79,80].

RS-XRF, PIXE and LA-ICP-MS are the only techniques that can been applied to correlate MR signal and local iron concentration voxel by voxel [41,71,81]. These techniques can be utilized as the gold standard for mapping the local iron tissue concentration.