**2.1.2 Special considerations for biological samples**

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 achieve both of these.

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 (McRae, Bagchi et al. 2009).

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