Sample preparation techniques for the observation of environmental biopolymers by atomic force microscopy

Although the structural characterization of environmental biopolymers is of prime importance in order to understand their roles in the natural environment, only limited analytical techniques are available that allow the determination of their morphologies and size distributions, due mainly to the complexity, heterogeneity and polydispersity of these organic colloids. Atomic force microscopy (AFM) has become an important technique for direct imaging of biomolecules with a sub nanometer resolution. This paper describes a systematic optimization of the AFM sample preparation technique prior to direct observation under ambient conditions. Three preparation methods (drop deposition, adsorption, ultracentrifugation) were tested on two reference compounds: a humic substance and a polysaccharide. The methods were evaluated using, where possible, quantitative analysis of the AFM images. The results indicated that each of the commonly used preparation techniques had potential artefacts, but when used in concert, structural parameters corresponding to those measured in solution could be determined. AFM height measurements performed under conditions of variable humidity demonstrated no observable structural effects on the biopolymers.