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Preparation of Biological Specimens

The X-ray Fluorescence Microprobe is an excellent tool for mapping trace metal distributions in biological systems at sub-micron length scales .  In particular, sensitivity is extremely high to first-row transition metals, i.e., K - Se, but also reaches down to Si.  Heavier metals can be imaged using L-shell [Cd-Pu] and M-shell [Os+] fluorescence.  It is therefore extremely important that the distribution of these metals not be disturbed during specimen preparation.

Most approaches to specimen preparation for metals mapping in animal tissues rely on metals immobilisation.  This can occur through chemical fixation or cryo-fixation followed by dehydration or freeze substitution.

A recent investigation [1] has indicated that formaldehyde fixing can result in metals redistribution in certain types of tissue sections, and that a short rinse in PBS can also disturb metals distribution.  In that article, cryofixation and drying is used as the reference standard for preserving metals distributions.

Unfortunately it is not always possible to plunge freeze a specimen, especially when the specimen is so large that cooling rates are not sufficient to prevent ice-crystal formation [2].  However, where plunge freezing and freeze-drying (or freeze substitution) is an option, we at least recommend that a comparison of the two techniques be performed as a part of the beamtime request.

Adherent cells can be grown directly onto silicon nitiride windows, and these are compatible with a variety of imaging and spectroscopic investigations [3].  We have a limited supply of these windows at the beamline; so please feel free to ask for some if you require less than 10 or so windows for your investigation.  However, if you need more than this or plan repeated trips you should consider these to be a part of your cost for the beamline access.  Such windows can be obtained from one of a number of companies including Silson.  Please feel free to consult us prior to making your purchase.

[1]  Hackett et al., Analyst 136, 2941 (2011).

[2]  Studer et al., Journal of Microscopy 203, 285 (2001).

[3]  Carter et al., Molecular Biosystems 6, 1316 (2010).