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 kHz 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. Keywords: atomic force microscopy (AFM); off-resonance tapping (ORT); pulsed-force mode; feedback contro

Piezoresistive AFM cantilevers surpassing standard optical beam deflection in low noise topography imaging

Optical beam deflection (OBD) is the most prevalent method for measuring cantilever deflections in atomic force microscopy (AFM), mainly due to its excellent noise performance. In contrast, piezoresistive strain-sensing techniques provide benefits over OBD in readout size and the ability to image in light-sensitive or opaque environments, but traditionally have worse noise performance. Miniaturisation of cantilevers, however, brings much greater benefit to the noise performance of piezoresistive sensing than to OBD. In this paper, we show both theoretically and experimentally that by using small-sized piezoresistive cantilevers, the AFM imaging noise equal or lower than the OBD readout noise is feasible, at standard scanning speeds and power dissipation. We demonstrate that with both readouts we achieve a system noise of ≈0.3 Å at 20 kHz measurement bandwidth. Finally, we show that small-sized piezoresistive cantilevers are well suited for piezoresistive nanoscale imaging of biological and solid state samples in air

Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy

The atomic force microscope has become an established research tool for imaging microorganisms with unprecedented resolution. However, its use in microbiology has been limited by the difficulty of proper bacterial immobilization. Here, we have developed a microfluidic device that solves the issue of bacterial immobilization for atomic force microscopy under physiological conditions. Our device is able to rapidly immobilize bacteria in well-defined positions and subsequently release the cells for quick sample exchange. The developed device also allows simultaneous fluorescence analysis to assess the bacterial viability during atomic force microscope imaging. We demonstrated the potential of our approach for the immobilization of rod-shaped Escherichia coli and Bacillus subtilis. Using our device, we observed buffer-dependent morphological changes of the bacterial envelope mediated by the antimicrobial peptide CM15. Our approach to bacterial immobilization makes sample preparation much simpler and more reliable, thereby accelerating atomic force microscopy studies at the single-cell level

High-frequency multimodal atomic force microscopy

Multifrequency atomic force microscopy imaging has been recently demonstrated as a powerful technique for quickly obtaining information about the mechanical properties of a sample. Combining this development with recent gains in imaging speed through small cantilevers holds the promise of a convenient, high-speed method for obtaining nanoscale topography as well as mechanical properties. Nevertheless, instrument bandwidth limitations on cantilever excitation and readout have restricted the ability of multifrequency techniques to fully benefit from small cantilevers. We present an approach for cantilever excitation and deflection readout with a bandwidth of 20 MHz, enabling multifrequency techniques extended beyond 2 MHz for obtaining materials contrast in liquid and air, as well as soft imaging of delicate biological samples

Design of a high-bandwidth tripod scanner for high speed atomic force microscopy

Tip-scanning high-speed atomic force microscopes (HS-AFMs) have several advantages over their sample-scanning counterparts. Firstly, they can be used on samples of almost arbitrary size since the high imaging bandwidth of the system is immune to the added mass of the sample and its holder. Depending on their layouts, they also enable the use of several tip-scanning HS-AFMs in combination. However, the need for tracking the cantilever with the readout laser makes designing tip-scanning HS-AFMs difficult. This often results in a reduced resonance frequency of the HS-AFM scanner, or a complex and large set of precision flexures. Here, we present a compact, simple HS-AFM designed for integrating the self-sensing cantilever into the tip-scanning configuration, so that the difficulty of tracking small cantilever by laser beam is avoided. The position of cantilever is placed to the end of whole structure, hence making the optical viewing of the cantilever possible. As the core component of proposed system, a high bandwidth tripod scanner is designed, with a scan size of 5.8 µm × 5.8 µm and a vertical travel range of 5.9 µm. The hysteresis of the piezoactuators in X- and Y-axes are linearized using input shaping technique. To reduce in-plane crosstalk and vibration-related dynamics, we implement both filters and compensators on a field programmable analog array. Based on these, images with 512 × 256 pixels are successfully obtained at scan rates up to 1024 lines/s, corresponding to a 4 mm/stip velocity

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

Studying biological membranes with extended range high-speed atomic force microscopy

High-speed atomic force microscopy has proven to be a valuable tool for the study of biomolecular systems at the nanoscale. Expanding its application to larger biological specimens such as membranes or cells has, however, proven difficult, often requiring fundamental changes in the AFM instrument. Here we show a way to utilize conventional AFM instrumentation with minor alterations to perform high-speed AFM imaging with a large scan range. Using a two-actuator design with adapted control systems, a 130 x 130 x 5 mu m scanner with nearly 100 kHz open-loop small-signal Z-bandwidth is implemented. This allows for high-speed imaging of biologically relevant samples as well as high-speed measurements of nanomechanical surface properties. We demonstrate the system performance by real-time imaging of the effect of charged polymer nanoparticles on the integrity of lipid membranes at high imaging speeds and peak force tapping measurements at 32 kHz peak force rate

A hybrid polymer/ceramic/semiconductor fabrication platform for high-sensitivity fluid-compatible MEMS devices with sealed integrated electronics

Active microelectromechanical systems can couple the nanomechanical domain with the electronic domain by integrating electronic sensing and actuation mechanisms into the micromechanical device. This enables very fast and sensitive measurements of force, acceleration, or the presence of biological analytes. In particular, strain sensors integrated onto MEMS cantilevers are widely used to transduce an applied force to an electrically measurable signal in applications like atomic force microscopy, mass sensing, or molecular detection. However, the high Young's moduli of traditional cantilever materials (silicon or silicon nitride) limit the thickness of the devices, and therefore the deflection sensitivity that can be obtained for a specific spring constant. Using softer materials such as polymers as the structural material of the MEMS device would overcome this problem. However, these materials are incompatible with high-temperature fabrication processes often required to fabricate high quality electronic strain sensors. We introduce a pioneering solution that seamlessly integrates the benefits of polymer MEMS technology with the remarkable sensitivity of strain sensors, even under high-temperature deposition conditions. Cantilevers made using this technology are inherently fluid compatible and have shown up to 6 times lower force noise than their conventional counterparts. We demonstrate the benefits and versatility of this polymer/ceramic/semiconductor multi-layer fabrication approach with the examples of self-sensing AFM cantilevers, and membrane surface stress sensors for biomolecule detection