Transitional-Tapping AFM: Probing Stochastic Molecular Dynamics at Sub-kBT Precision

Conventional tapping-mode atomic force microscopy (AFM) is limited in its ability to resolve time-varying interaction forces during rapid and stochastic molecular transitions at a sample pixel while scanning. Resolving these transitions is essential for tracking molecular assembly and rearrangements in real-time. We introduce Transitional-tapping ( Tt ) AFM, a technique that achieves this by directly measuring sample stochasticity from higher harmonic tip interaction forces. Tt-AFM harmonizes tip-sample motion by fine-tuning tip oscillations to sustain continuous engagement during scanning. We demonstrate its capability by uncovering transient native-like protein intermediates, that align remarkably with AlphaFold and Protein Data Bank predictions. The imaged intermediates retain native folds but undergo subtle chain rearrangements, persisting as ‘rest-stops’ for nanoseconds along the folding landscape to stabilize. Tip dynamics reveal that shear-thinning guides folding through sub-diffusive Lévy-jump kinetics. Transitional-tapping fundamentally transforms AFM’s capability to probe stochasticity and intermediate energetics at sub-k _B T precision, unlocking new frontiers in soft-matter AFM.