Reconstructing Multiwell Potentials with Steep Gradients Using Stochastically Excited Spring Probes

Measurements of free energy landscapes are critical for understanding the basis of many physical, chemical, and biological interactions. Statistical mechanics provide exact equations to calculate free energies, but are built on the assumption that all possible configurations of the system are sampled. The most pronounced limit to accurate free energy computations is therefore the imperfect sampling of a potential field, particularly in the case of interactions with steep gradients and short reaction coordinates. We show through simulations that increasing the stochastic fluctuations of a harmonic probe by active excitation results in increased sampling times of high gradient adhesive interactions and leads to the reconstruction of a more accurate energy landscape. We use Brownian dynamics simulations to test the impact of probe approach velocity, stiffness, and thermal energy to sample complex energy landscapes with multiple wells of various depths and slopes to understand the accuracy of energy surface reconstruction. We then show experimentally that through the application of optimal stochastic excitations, we are able to obtain accurate energy landscape reconstructions for different probe and landscape parameters due to improved sampling of previously poorly probed interactions