High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Enhanced feedback performance in off-resonance AFM modes through pulse train sampling.

Dynamic atomic force microscopy (AFM) modes that operate at frequencies far away from the resonance frequency of the cantilever (off-resonance tapping (ORT) modes) can provide high-resolution imaging of a wide range of sample types, including biological samples, soft polymers, and hard materials. These modes offer precise and stable control of vertical force, as well as reduced lateral force. Simultaneously, they enable mechanical property mapping of the sample. However, ORT modes have an intrinsic drawback: a low scan speed due to the limited ORT rate, generally in the low-kilohertz range. Here, we analyze how the conventional ORT control method limits the topography tracking quality and hence the imaging speed. The closed-loop controller in conventional ORT restricts the sampling rate to the ORT rate and introduces a large closed-loop delay. We present an alternative ORT control method in which the closed-loop controller samples and tracks the vertical force changes during a defined time window of the tip-sample interaction. Through this, we use multiple samples in the proximity of the maximum force for the feedback loop, rather than only one sample at the maximum force instant. This method leads to improved topography tracking at a given ORT rate and therefore enables higher scan rates while refining the mechanical property mapping.

A high-bandwidth voltage amplifier for driving piezoelectric actuators in high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) is a technique capable of revealing the dynamics of biomolecules and living organisms at the nanoscale with a remarkable temporal resolution. The phase delay in the feedback loop dictates the achievable speed of HS-AFM instruments that rely on fast nanopositioners operated predominantly in conjunction with piezoelectric actuators (PEAs). The high capacitance and high operating voltage of PEAs make them difficult to drive. The limited bandwidth of associated high-voltage piezo-amplifiers is one of the bottlenecks to higher scan speeds. In this study, we report a high-voltage, wideband voltage amplifier comprised of a separate amplification and novel voltage-follower power stage, requiring no global feedback. The reported amplifier can deliver a current over ±2 amps, offers a small-signal bandwidth of 1 MHz, and exhibits an exceptionally low phase lag, making it particularly well suited for the needs of next-generation HS-AFMs. We demonstrate its capabilities by reporting its achievable bandwidth under various PEA loads and showcasing its merit for HS-AFM by imaging tubulin protofilament dynamics at sub-second frame rates.

An atomic force microscope integrated with a helium ion microscope for correlative nanoscale characterization.

In this work, we report on the integration of an atomic force microscope (AFM) into a helium ion microscope (HIM). The HIM is a powerful instrument, capable of imaging and machining of nanoscale structures with sub-nanometer resolution, while the AFM is a well-established versatile tool for multiparametric nanoscale characterization. Combining the two techniques opens the way for unprecedented in situ correlative analysis at the nanoscale. Nanomachining and analysis can be performed without contamination of the sample and environmental changes between processing steps. The practicality of the resulting tool lies in the complementarity of the two techniques. The AFM offers not only true 3D topography maps, something the HIM can only provide in an indirect way, but also allows for nanomechanical property mapping, as well as for electrical and magnetic characterization of the sample after focused ion beam materials modification with the HIM. The experimental setup is described and evaluated through a series of correlative experiments, demonstrating the feasibility of the integration.

Integration of sharp silicon nitride tips into high-speed SU8 cantilevers in a batch fabrication process.

Employing polymer cantilevers has shown to outperform using their silicon or silicon nitride analogues concerning the imaging speed of atomic force microscopy (AFM) in tapping mode (intermittent contact mode with amplitude modulation) by up to one order of magnitude. However, tips of the cantilever made out of a polymer material do not meet the requirements for tip sharpness and durability. Combining the high imaging bandwidth of polymer cantilevers with making sharp and wear-resistant tips is essential for a future adoption of polymer cantilevers in routine AFM use. In this work, we have developed a batch fabrication process to integrate silicon nitride tips with an average tip radius of 9 ± 2 nm into high-speed SU8 cantilevers. Key aspects of the process are the mechanical anchoring of a moulded silicon nitride tip and a two-step release process. The fabrication recipe can be adjusted to any photo-processable polymer cantilever.

High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6.

The self-assembly of protein complexes is at the core of many fundamental biological processes1, ranging from the polymerization of cytoskeletal elements, such as microtubules2, to viral capsid formation and organelle assembly3. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement. As self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy (HS-AFM) due to the non-negligible tip-sample interaction forces involved in current methods. We have developed a HS-AFM technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. 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 the formation of the centriole organelle. Our analysis reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical structure.