Simultaneous topography and recognition imaging: physical aspects and optimal imaging conditions

Abstract

Simultaneous topography and recognition imaging (TREC) allows for the investigation of receptor distributions on natural biological surfaces under physiological conditions. Based on atomic force microscopy (AFM) in combination with a cantilever tip carrying a ligand molecule, it enables us to sense topography and recognition of receptor molecules simultaneously with nanometre accuracy. In this study we introduce optimized handling conditions and investigate the physical properties of the cantilever-tip-sample ensemble, which is essential for the interpretation of the experimental data gained from this technique. In contrast to conventional AFM methods, TREC is based on a more sophisticated feedback loop, which enables us to discriminate topographical contributions from recognition events in the AFM cantilever motion. The features of this feedback loop were investigated through a detailed analysis of the topography and recognition data obtained on a model protein system. Single avidin molecules immobilized on a mica substrate were imaged with an AFM tip functionalized with a biotinylated IgG. A simple procedure for adjusting the optimal amplitude for TREC imaging is described by exploiting the sharp localization of the TREC signal within a small range of oscillation amplitudes. This procedure can also be used for proving the specificity of the detected receptor-ligand interactions. For understanding and eliminating topographical crosstalk in the recognition images we developed a simple theoretical model, which nicely explains its origin and its dependence on the excitation frequency.

Topography and Recognition Imaging of Protein‐Patterned Surfaces Generated by AFM Nanolithography

“…However, the strength of the recognition signal from full-amplitude feedback was about 1.6 times that of half-amplitude feedback. These results show a similar tendency to that observed in our recent studies [16] on single molecules.…”

AFM functional imaging on vascular endothelial cells

“…Since polyethylene glycol (PEG) chains between AFM tips and ligand molecules are particularly advantageous for use in molecular recognition force spectroscopy (MRFS) (Hinterdorfer and Dufrêne, 2006), they are essentially necessary for TREC measurements (Preiner et al, 2009).…”

“…The set-point amplitude is adjusted to a value close to the free amplitude. The free amplitude is adjusted to be less that the extended PEG-linker to provide the proper recognition image with high efficiencies and repeatability (Chtcheglova et al, 2007;Preiner et al, 2009). When such a tip-tethered ligand binds to its receptor on the sample surface (i.e.…”

“…In contrast to the conventional MAC mode, which exploits a peak-to-peak value of oscillating amplitude (full amplitude) as a feedback parameter, TREC uses the lower part of the oscillation (called as half-amplitude feedback) to drive a feedback loop for obtaining the topography image, whereas the upper part of the oscillation is used for generation of the recognition image (Figure 4). Moreover, using such sophisticated feedback allows accurate determination of the surface topography (Preiner et al, 2009). To provide more details, the time-resolved deflection signal of the oscillating cantilever is low-pass filtered to remove the thermal noise, the DC (direct current) is offset leveled and amplified before splitting into the lower (U down ) and upper (U up ) parts of the oscillations.…”

“…Subsequent peaks form a staircase function, which is then filtered and fed into the AFM controller, where U down drives the feedback loop to record the topography image and U up provides the information to establish the corresponding recognition image. Moreover, the utilization of cantilevers with low Q factor ($ 1 in liquid) in combination with a proper chosen driving frequency and amplitude regime enables the that both types of information are unrelated (Ebner et al, 2005;Preiner et al, 2009). …”