In AFM-based single molecule force spectroscopy, it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Although this assumption may hold for flexible, compact molecules, studies have shown that it may not be appropriate for fairly rigid molecules, where measured forces can be a fraction of the actual values experienced by the molecule. Because the pulling geometry can substantially influence the values measured by the AFM, we investigate a method to minimize the pulling angle prior to conducting a pulling experiment. The method presented herein uses small circular movements to locate the molecule’s substrate attachment site and reposition the cantilever. By using data gathered from a previous study, we were able to repeatedly align a molecule’s attachment sites via simulation of the program, thereby demonstrating the effectiveness of the alignment method.

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