In this work we have designed a simple system to investigate empirically the image contrast of tapping-mode atomic force microscopy (TMAFM). We modified the cantilever tips with protein molecules (bovine serum albumin or goat anti-biotin antibody) and used these protein-modified cantilevers to scan poly-L-lysine films and antibody layers deposited on mica in air under ambient conditions. We also investigated the effects of manipulating the setpoint voltage in this system. It was found that extra topographic features with a patchlike appearance were introduced into the TMAFM images of both the poly-L-lysine and antibody films when scanned with the protein-modified tips, even at initial preset setpoints, and were superimposed on the topography of the samples. The surface coverage of the patchlike features in the TMAFM images changes significantly with the setpoint voltage in a reversible and nonlinear manner. These are believed to arise from the surface indentation of the sample or from the structural deformation of the proteins at the tip induced in TMAFM imaging. Interestingly, it was observed in the experiment that no structural alteration or damage was discernible on the sample surface, even after continuous scanning with the protein-modified tips for a long period of time, with varying setpoint voltage. This study provides experimental evidence that cantilever tips modified with protein molecules or, under certain circumstances, even unmodified tips introduce extra topographical features (i.e., artifacts) and enhance the image contrast of TMAFM imaging of soft materials, which is dependent on their mechanical properties.
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