Several studies have shown that heterogeneous agents are able to reduce required starting protein concentration and induction time for crystallization. However, in many cases, the molecular mechanism by which heterogeneous agents promote the nucleation is still unclear, although examples of epitaxial crystallization have been reported. Here, a set of focused crystallization trials, together with a review of the literature data, point out that the process of heterogeneous nucleation can be described by a nonclassical mechanism. We suppose that due to concentration fluctuations, unstable prenucleation aggregates homogeneously form in solution in conditions of low supersaturation, when the chemistry of solution favors this event. Then heterogeneous substrates stabilize prenucleation clusters by nonspecific interactions and trigger the nucleation event. This concept applied to proteins, but also to any colloidal system where the ordered assembly is governed by weak interactions, highlights the importance of the chemistry of the solution as a key parameter to achieve nucleation, even in the presence of heterogeneous nucleants.

We present an overview of recent advances in the application of Fourier Transform Infrared (FTIR) microscopy for analysis of complex, multicomponent, and multilayer samples such as those typically encountered in the field of heritage materials. This technique is particularly useful since it allows identification and localization of both organic and inorganic (if IR active) compounds. New improvements have been possible thanks to the introduction of ad hoc sample preparation methods to obtain either thin or cross sections that allow both avoidance of contamination from organic embedding resin and improvement of the quality of the acquired spectra. Moreover, integrated use of spectra registered in the near-infrared (NIR) and mid-infrared (MIR) regions allows better comprehension of cross section composition. Data interpretation has been improved thanks to the development of chemometric methods for elaboration of hyperspectral data. A new and very promising field is the development of enhanced FTIR methods for detection of trace components in microextracts. These systems, allowing detection of extractable organic compounds from about 0.1 mg of sample, will be extremely useful in the future for analysis of natural and synthetic colorants, varnishes extracted, for instance, from cotton swabs used during cleaning of paintings, and organic residues on archeological remains.

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