Application notes

The possibility of atomic-scale imaging with atomic force microscopy (AFM) is an exciting discovery. But achieving good image resolution with an AFM is a very complicated issue. First, it depends on the apex diameter of probe tip used (typically of the order of 20 nm). The latter mainly determines the tip-sample interaction area. In contact mode, images of crystal surfaces show atomic-scale patterns similar to a crystallographic atomic arrangement. The absence of atom-size defects in such images means that the tip-sample contact area is considerably larger than the atomic size. However, it is small enough to reproduce periodic micro-variations of interaction force corresponding to the surface lattice when the tip slides on it. In tapping mode, the intermittent tip–sample contact allows gentler imaging with forces that can be much lower than in contact mode. So tapping mode has a significantly wider range of applications as it can be used to image many soft samples without deformation or destruction. At present, tapping mode applications in air are most common even though its image resolution (~1nm) is not as high as in contact mode. But the resolution in tapping mode is higher than the apex size. This is most likely related to the fact that the tip-sample contact area depends not only on the tip size but also on the interaction force (the weaker the force the better resolution) and the mechanical properties of the sample and the tip. But for many applications this resolution is not always enough. For instance, the need for higher resolution is one of the most complicated problems associated with AFM imaging of biomolecules. Especially when the internal structure of the molecules is of interest. The relatively large apex radius of commercially available Si AFM probes (5–10 nm) does not allow the imaging of the internal molecular features (for example DNA). It is desirable to improve resolution by using sharp probes. This can be done with HRC probes, whose development has made imaging of soft fragile samples with molecular-scale resolution much more achievable. In particular HRC could serve as a very useful tool for the investigation of various biopolymers.

Recommendations of using HRC

The HRC has a thin long spike with a high aspect ratio, is very fragile and can be easy damaged (especially for example during engagement process). To achieve the highest image quality with the highest resolution the forces between a sample and a tip should be very weak. If HRC is used to image soft samples large interrelated forces could cause sample damage as a sharper tip means higher pressure within the contact area. Therefore, using soft cantilevers with stiffness of the order of few N/m is preferable (soft cantilevers with stiffness of the order of few tenth of N/m may cause jump-to-contact instability). Careful engagement procedure to avoid the tip damage is strongly recommended. A light tapping constant force mode should be used. (The free oscillation amplitude should be of the order of few nm, about 4 nm is recommended, the set-point amplitude should be close to the free-oscillating amplitude, about 98% is recommended.) Scanning rate of few Hz is recommended and the start scan size should be of the order of a few tens of nm. RMS roughness of the surface to be investigated should be below 20nm. In general, there are additional smaller tips near the main one. Though the extra tips are of different height and angle, unwanted double imaging is possible on rough surfaces with roughness larger than 20nm. Additionally scanning of rough and hard surfaces (e.g. silicon surface) or using a high scanning rate could also damage the tip or a sample.

Case studies

Single-stranded DNA

Double-stranded DNA

Single- and double-stranded DNA on mica and HOPG

Synthesis of triple-stranded DNA

Dodecylamine lamellar structures

Polydiacetylene crystal

Galacturonan molecules

Virus particles