Unraveling Molecular Dynamics with High-Speed Video-Rate Atomic Force Microscopy


Dimitar R. Stamov,* Tanja Neumann, Andreas Kraus, André Körnig, Tilo Jankowski,


Detlef Knebel, Torsten Jähnke, Thomas Henze, and Heiko Haschke JPK BioAFM, Bruker Nano Surfaces, Am Studio 2D, 12489 Berlin, Germany *dimitar.stamov@bruker.com


Abstract: This article describes the application of the fastest commercially available video-rate atomic force microscope (AFM) for studying the molecular dynamics of biological samples with a temporal resolution reaching 20 ms per frame. High-speed AFM was used for monitoring the kinetics of the temperature-induced ripple phase transition in supported lipid bilayers, DNA origami streptavidin-biotin binding, thermodynamic rehybridization of DNA, collagen type I fibrillogenesis, and the mobile annexin V trimer dynamics in 2D protein crystals. With no further need for sample processing, the applied method offers a breakthrough in molecu- lar and sample dynamics measurements by enabling the real-time visualization of molecular processes and structural transitions at near-native conditions.


Keywords: high-speed AFM, collagen I, DNA origami, annexin V, DMPC ripple phases


Introduction Atomic force microscopy (AFM) is a powerful tool that


allows the comprehensive study of mechanical properties and interactions with nanometer resolution. Te last three decades have established the technique as the instrument of choice for high-end structural analysis of samples ranging from single molecules to complex biological systems, such as proteins and cells. Unlike other high-resolution imaging techniques, such as super-resolution light and advanced elec- tron microscopy, AFM does not require any sample label- ing or modification. In turn, that means that no preparation artifacts are introduced during imaging, thus enabling the capacity to study the specimens at their near-physiological or native state/environment. High-resolution imaging techniques, such as X-ray crystal-


lography, transmission electron microscopy, as well as conven- tional AFM, have contributed to our in-depth understanding of various biological systems. Nevertheless, they have struggled with the functional interpretation of molecular and dynamic processes taking place in living systems, by only partially addressing temporal snapshots of specific biological events and processes. Studying single macromolecule dynamics and the function of complex biological systems, such as individual liv- ing cells, requires a tool that can provide both high spatial and high temporal resolution. High-speed AFM has come a long way since the first


attempts to visualize biomolecular dynamics [1], or since the first postulated theoretical considerations for the scan speed limitations in conventional AFM [2]. Developments in the last 15–20 years have made possible the application of ultra-short cantilevers, high-resonance piezoactuator-based sample scan- ners, feedback systems, and optical beam deflection detectors that have ultimately enabled the studies of dynamic biomo- lecular processes with high-speed prototypes [3,4], as well as


10 doi:10.1017/S155192952200061X


commercial systems enabling both tip [5,6] as well as sample- scanning acquisition rates of 10–20 frames per second [7]. Tis has effectively been made possible by the much higher oscil- lation frequencies of the used cantilevers, improved feedback bandwidths, as well as enhanced XY-movement, which in turn enable higher scanning rates without decreasing the resolution of the measurement, or damaging very sensitive samples, as reviewed in [8,9]. We have recently developed and launched the NanoRacer®


,


which is currently the fastest commercial high-speed AFM, able to operate at a video-rate scanning speed of 50 frames/sec. Te instrument features 3-axis closed-loop sample scanning, high detector bandwidth of 8 MHz, novel high-speed power amplifier, as well as advanced algorithms for scanner control and feedback loop error correction, such as iterative inversion- based adaptive scanning and harmonic motion to improve feedback loop bandwidth [10]. Tis is further complemented by the use of dynamic proportional-integral-derivative (PID) control, minimizing feedback saturation errors. In combina- tion with a small excitation laser spot size, this enables the high-speed stable successive imaging of soſt and fragile bio- logical specimens with a temporal resolution of near-20 ms. Te NanoRacer was a recipient of the Microscopy Today 2021 Innovation Award based on its usefulness to the microscopy community [10]. In this article we demonstrate how high-speed AFM can


be applied for studying single-molecule dynamics in biologi- cal systems, for example, studying lipid ripple phase formation, thermodynamic DNA rehybridization, collagen type I fibril formation, as well as annexin V (A5) rotational dynamics.


Methods and Materials Sample preparation. All measurements were carried out


in a customized fluid chamber and optional temperature con- trol including a muscovite mica disk as a support. To expose the atomically flat mica surface, the substrates were freshly cleaved prior to sample preparation. DMPC lipid bilayers. 150 μM 1,2-dimyristoyl-sn-glycero-


3-phosphocholine (DMPC) solutions were prepared by dis- solving DMPC (Avanti Lipids) in sample buffer (150 mM NaCl, 20 mM Tris, 5 mM EDTA buffer (pH 7.6)). Te protocol included depositing 5 μL of DMPC solution to the substrate, adding 1 μL of 100 mM CaCl2


, followed by 45 min incubation


at 4°C. Aſter 2-fold rinsing in sample buffer, the specimens were immediately imaged. DNA origami nanostructures (DONs). DONs were pro-


vided by collaborators [11] and prepared as previously described [12]. Each rectangular origami template carried five covalently bound biotin residues. 10 μL of 1 nM of DNA origami solution


www.microscopy-today.com • 2022 May


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