Invited Paper NS+MN+PC+SS-WeA9
Open-hardware, High-speed Atomic Force Microscopy using Photothermal Off-resonance Tapping

Wednesday, October 24, 2018, 5:00 pm, Room 203A

Self-assembly of protein complexes is at the core of many fundamental biological processes. To reach a comprehensive understanding of the underlying protein self-assembly reactions, high spatial and temporal resolution must be attained. This is complicated by the need to not interfere with the reaction during the measurement. Since self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy due to the non-negligible tip-sample interaction forces involved in current methods. Here we develop a high-speed atomic force microscopy technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. Using photothermal actuation on ultra-small HS-AFM cantilevers we perform force-distance curves at two orders of magnitude higher rates than in conventional off-resonance methods. From the time-domain tip sample interaction we extract tip-sample force curves to quantify the “static”forces due to the cantilever deflection, and the “impact” forces due to the rapid deceleration of the cantilever tip upon impact. Experimental characterization of the tip-sample forces in HS-tapping mode-AFM and PORT revealed that imaging forces in PORT are less than 1/5^thof those exerted in conventional HS-AFM.

One of the key enabling factors for PORT is the real time control of the cantilever position using photothermal excitation. This requires low level, control of the feedback architecture and optimized AFM instrumentation. I will discuss the relevant components developed in my laboratory and explain how we share them with the broader scientific community using an open-hardware scheme.

We apply PORT to dissect the self-assembly reaction of SAS-6 proteins, which form a nine-fold radially symmetric ring-containing structure that seeds formation of the centriole organelle present in all eukaryotic cells. Using machine learning algorithms we traced hundreds of molecules over time to extract reaction kinetics from single molecule interactions. These measurements show that 9-fold closed SAS-6 rings are under pre-tension in their natural state. Due to the high temporal and force resolution provided by PORT, we found that, contrary to the current belief, more than one assembly route exists to reach the nine fold symmetry. These observation resets our current thinking about the assembly kinetics of this crucial step in cell replication.