AFM imaging

AFM analysis was performed in air, at room temperature, in the dynamic mode with a Nanoscope IIIA (Veeco) and an Agilent 5500 PicoPlus (Agilent Technologies) microscopes. Tapping mode AFM was carried out using silicon cantilevers with nominal curvature radius of 8 nm (Bruker), nominal force constant of 4 N/m and resonance frequency in the 50–80 kHz range. The set-points used were kept in the 0.3–0.6 V range, while the free amplitude values were in the 0.7–0.8 V range. The images (from 512 × 512 up to 2048 × 2048 pixels) were recorded at a scan rate of 1 line/s. A minimum of three independent samples of each viroid preparation at each buffer composition (nine in total) were used, thus rendering more than 27 samples analyzed by AFM, from which different AFM fields were imaged.

The influence of the tip radius is a relevant issue when imaging nanometer structures by AFM. In principle, with a nominal tip radius of 8 nm (which is considered the optimal one for imaging biological samples in air using tapping mode, and consequently selected for this work), distances below this value are difficult to resolve. Furthermore, for larger distances tip convolution is still present and leads to a widening of the imaged structure. This later aspect has indeed been taken into account here, since the length distances have been measured between points located at half of the height of the imaged structure (as an example, see graphs in ^{Figs. 1}–³ and ⁵–¹⁰). This procedure leaves out of the measurement approximately 8 nm (the tip nominal radius) of lateral extension at each of both extremes of the imaged molecule, thus minimizing the tip size effects.