In atomic force microscopy (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. Previously, we have proposed a method to align a molecule’s substrate and cantilever attachment sites and tested it in a simulated environment. Here we continue our work and test the alignment program in an experimental environment. In this paper we demonstrate that circling-induced force fluctuations are the result of stretching and relaxing a tethered molecule and we present the results of an alignment trial. Combined, these preliminary results demonstrate the feasibility of the alignment program and are a promising step towards correcting pulling geometry errors in single molecule force spectroscopy studies.

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