7. Sample preparation
7.1 Introduction
The SPM has permitted an unprecedented level of exploration of the effects of sample preparation on the conformation of biomolecules. This is because of the ease with which samples are prepared compared to electron microscopy and also because of the freedom to image under solution. Development will undoubtedly continue apace, but we illustrate some of the issues with studies of the deposition of DNA on mica.
7.2 Adsorption from solution.
Rivettii, Guthold and Bustamante (Rivetti, Guthold et al. 1996) have undertaken a careful study of the end-to-end length of DNA molecules as a function of the sample preparation conditions. They showed that, in the absence of stirring, the number of molecules adsorbed onto the surface,nF(t) at a time t varies with the initial concentration, n0, and time as expected for translational diffusion to the surface followed by irreversible adsorption, for which (Lang and Coates 1968)
where D is the translational diffusion coefficient for the molecular weight of molecule being deposited.
The chemistry of the substrate is also important. This is because adsorption onto the surface is a replacement reaction: Surface water must be replaced, and the free-energy cost of doing so must be favorable. Thus hydrophobic molecules will rapidly displace water at a hydrophobic surface. Adsorption onto a highly hydrophilic surface is more difficult. Mica is commonly used as a substrate and it is (nominally) quite hydrophilic, so perhaps contamination plays a role in reducing this hydrophilicity when mica is cleaved in laboratory air.
