Simultaneous topography and recognition imaging: physical aspects and optimal imagingconditions

Abstract

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