Simultaneous Atomic Force Microscopy Research Articles

Calibration of the oscillation amplitude of quartz tuning fork-based force sensors with astigmatic displacement microscopy.

Quartz tuning forks and qPlus-based force sensors offer an alternative approach to silicon cantilevers for investigating tip-sample interactions in scanning probe microscopy. The high-quality factor (Q) and stiffness of these sensors prevent the tip from jumping to the contact, even at sub-nanometer amplitude. The qPlus configuration enables simultaneous scanning tunneling microscopy and atomic force microscopy, achieving spatial resolution and spectroscopy at the subatomic level. However, to enable precise measurement of tip-sample interaction forces, confidence in these measurements is contingent upon the accurate calibration of the spring constant and oscillation amplitude of the sensor. Here, we have developed a method called astigmatic displacement microscopy with picometer sensitivity.

Electrochemical AFM/STM with a qPlus sensor: A versatile tool to study solid-liquid interfaces.

Atomic force microscopy (AFM) that can be simultaneously performed with scanning tunneling microscopy (STM) using metallic tips attached to self-sensing quartz cantilevers (qPlus sensors) has advanced the field of surface science by allowing for unprecedented spatial resolution under ultrahigh vacuum conditions. Performing simultaneous AFM and STM with atomic resolution in an electrochemical cell offers new possibilities to locally image both the vertical layering of the interfacial water and the lateral structure of the electrochemical interfaces. Here, a combined AFM/STM instrument realized with a qPlus sensor and a home-built potentiostat for electrochemical applications is presented. We demonstrate its potential by simultaneously imaging graphite with atomic resolution in acidic electrolytes. Additionally, we show its capability to precisely measure the interfacial solvent layering along the surface normal as a function of the applied potential.

Correlation between Single-Photon Emission and Size of Cesium Lead Bromide Perovskite Nanocrystals.

Emission photon statistics of semiconductor nanocrystal quantum dots (QDs), including lead halide perovskite nanocrystals (PNCs), are important fundamental and practical optical properties. Single QDs exhibit high-probability single-photon emission owing to the efficient Auger recombination between generated excitons. Because the recombination rate depends on QD size, single-photon emission probability should be size-dependent. Previous studies have researched QDs smaller than their exciton Bohr diameters (twice the Bohr radius of excitons). Here, we investigated the relationship between the single-photon emission behavior and size of CsPbBr3 PNCs to elucidate their size threshold. Simultaneous single-nanocrystal spectroscopy and atomic force microscopy observations on single PNCs with approximately 5-25 nm edge length showed that those smaller than approximately 10 nm, which had size-dependent photoluminescence (PL) spectral shifts, exhibited high-probability single-photon emissions, which decreased linearly with PNC volume. Novel single-photon emission, size, and PL peak correlations of PNCs are important for understanding the relationship between single-photon emission and quantum confinement.

Coexisting Ordered and Disordered Membrane Phases Have Distinct Modes of Interaction with Disease-Associated Oligomers.

Ordered membrane domains are thought to influence the attachment and insertion of toxic amyloid oligomers, and consequently, their toxicity. However, if and how the molecular aspects of this interaction depend on the membrane order is poorly understood. Here we measure the affinity, location, and degree of insertion of the small oligomers of hIAPP (human Islet Amyloid Polypeptide, associated with Type II diabetes) at near-physiological concentrations to adjacent domains of a biphasic lipid bilayer. Using simultaneous atomic force, confocal and fluorescence lifetime microscopy (AFM-FLIM), we find that hIAPP oligomers have a nearly 8-fold higher affinity to the disordered domains over the ordered domains. To probe whether this difference indicates different modes of interaction, we measure the change of lifetime of peptide-attached fluorescent labels induced by soluble fluorescence quenchers and also measure the kinetics of localized photobleaching. We find that in the raft-like ordered domains, the oligomers primarily lie on the aqueous interface with limited membrane penetration. However, in the neighboring disordered domains, their C-termini penetrate deeper into the lipid bilayer. We conclude that local membrane order determines not only the affinity but also the mode of interaction of amyloid oligomers, which may have significant implications for disease mechanisms.

Ionically Mediated Mechanical Deformation Associated with Memristive Switching

AbstractIonically mediated phenomena underpin the functioning of various devices, including batteries, solid oxide fuel cells, memristors, and neuromorphic devices. The ionic behavior corresponding to ionically mediated phenomena causes not only variations in the electrical properties but also mechanical deformation, which is crucial for device reliability. However, the interrelation between ionically mediated electrical properties and mechanical deformation has not been elucidated yet. This study investigates ionically mediated mechanical deformation accompanied by memristive switching in a TiO2 single crystal through simultaneous conductive atomic force microscopy and electrochemical strain microscopy. A comprehensive analysis indicates the existence of a relationship between mechanical deformation and memristive switching based on the ionic behavior. Furthermore, an ionic state variable is used to simplify the interrelation between the electrochemical strain hysteresis and memristive switching associated with applied voltage. This study provides insights on the ionic behavior and can be extended to other systems for the general analysis of the relationship between mechanical deformation and electrical properties.

In Situ Reproducible Sharp Tips for Atomic Force Microscopy

Atomically sharp tips are a requirement for scanning-probe microscopy, such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). Compared with STM, AFM imaging is more sensitive to the sharpness of tip apices because long-range forces act as a background signal on the high-resolution AFM images originating from short-range forces. Here we report the investigation of in situ reproducible sharp tips for AFM. We make an ${\mathrm{Ag}}_{2}\mathrm{S}$ crystal, a mixed ionic and electronic conductor, on a conventional $\mathrm{Si}$ cantilever, and controllably grow and shrink the $\mathrm{Ag}$ nanoprotrusion by changing the polarity of the bias voltage between the tip and the sample. We are able to reduce the contribution of long-range forces by growing a $\mathrm{Ag}$ nanoprotrusion on the ${\mathrm{Ag}}_{2}\mathrm{S}$ tip, and obtain atomic-resolution AFM images. We also confirm that the ${\mathrm{Ag}}_{2}\mathrm{S}$ tip with a $\mathrm{Ag}$ nanoprotrusion, the end of which presumably terminates in $\mathrm{Si}$ atoms, is capable of simultaneous AFM and STM measurements.

Robust scan synchronized force-fluorescence imaging

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells. To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe “pokes” the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Multi-Channel Exploration of O Adatom on TiO2(110) Surface by Scanning Probe Microscopy.

We studied the O2 dissociated state under the different O2 exposed temperatures with atomic resolution by scanning probe microscopy (SPM) and imaged the O adatom by simultaneous atomic force microscopy (AFM)/scanning tunneling microscopy (STM). The effect of AFM operation mode on O adatom contrast was investigated, and the interaction of O adatom and the subsurface defect was observed by AFM/STM. Multi-channel exploration was performed to investigate the charge transfer between the adsorbed O and the TiO2(110) by obtaining the frequency shift, tunneling current and local contact potential difference at an atomic scale. The tunneling current image showed the difference of the tunneling possibility on the single O adatom and paired O adatoms, and the local contact potential difference distribution of the O-TiO2(110) surface institutively revealed the charge transfer from TiO2(110) surface to O adatom. The experimental results are expected to be helpful in investigating surface/interface properties by SPM.

Complementary SEM-AFM of Swelling Bi-Fe-O Film on HOPG Substrate

The objective of this work is to study the delamination of bismuth ferrite prepared by atomic layer deposition on highly oriented pyrolytic graphite (HOPG) substrate. The samples’ structures and compositions are provided by XPS, secondary ion mass spectrometry (SIMS) and Raman spectroscopy. The resulting films demonstrate buckling and delamination from the substrates. The composition inside the resulting bubbles is in a gaseous state. It contains the reaction products captured on the surface during the deposition of the film. The topography of Bi-Fe-O thin films was studied in vacuum and under atmospheric conditions using simultaneous SEM and atomic force microscopy (AFM). Besides complementary advanced imaging, a correlative SEM-AFM analysis provides the possibility of testing the mechanical properties by using a variation of pressure. In this work, the possibility of studying the surface tension of the thin films using a joint SEM-AFM analysis is shown.

Mechanically Induced Conformation Change, Fluorescence Modulation, and Mechanically Assisted Photodegradation in Single Nanoparticles of the Conjugated Polymer Poly(9,9-dioctylfluorene).

We explored the possibility of nanoscale mechanical manipulation and control of photophysical properties of conjugated polymer nanoparticles. We carried out a simultaneous atomic force microscopy (AFM) and fluorescence microspectroscopy study on single nanoparticles of the conjugated polymer poly(9,9-dioctylfluorene). The nanoparticles are prepared by a reprecipitation method and have an average height of 27 nm, and their emission is dominated by the well-ordered β-phase conformation. Fluorescence polarization anisotropy and numerical simulations show that each particle contains at least three partly oriented straight β-phase segments surrounded by amorphous glass-phase polyfluorene chains. In the simultaneous experiments, an AFM tip was used to apply external force on a single nanoparticle, and a confocal fluorescence microscope was used to monitor in real time the resulting changes in the fluorescence intensity and spectra. In a nitrogen atmosphere, weak to moderate force of up to 1 μN acts mainly on the glass-phase polyfluorene chains by forming quenchers that cause an efficient and reversible fluorescence decrease, whereas the β-phase segments stay unaffected. A higher force of 5 μN, on the contrary, breaks the β-phase segments into multiple glass-phase segments, causing a net increase in fluorescence intensity. Under ambient air conditions, even a moderate force of 1 μN strongly accelerates the degradation of the nanoparticle by preferably photobleaching the β-phase and partially transforming it into the glass phase. These results will contribute to the fundamental knowledge on the relationship between photophysical and structural properties of polyfluorene nanostructures, and will also provide important feedback for potential applications of such nanostructures in flexible optoelectronic devices.

The qPlus sensor, a powerful core for the atomic force microscope.

Atomic force microscopy (AFM) was introduced in 1986 and has since made its way into surface science, nanoscience, chemistry, biology, and material science as an imaging and manipulating tool with a rising number of applications. AFM can be employed in ambient and liquid environments as well as in vacuum and at low and ultralow temperatures. The technique is an offspring of scanning tunneling microscopy (STM), where the tunneling tip of the STM is replaced by using a force sensor with an attached tip. Measuring the tiny chemical forces that act between the tip and the sample is more difficult than measuring the tunneling current in STM. Therefore, even 30 years after the introduction of AFM, progress in instrumentation is substantial. Here, we focus on the core of the AFM, the force sensor with its tip and detection mechanism. Initially, force sensors were mainly micro-machined silicon cantilevers, mainly using optical methods to detect their deflection. The qPlus sensor, originally based on a quartz tuning fork and now custom built from quartz, is self-sensing by utilizing the piezoelectricity of quartz. The qPlus sensor allows us to perform STM and AFM in parallel, and the spatial resolution of its AFM channel has reached the subatomic level, exceeding the resolution of STM. Frequency modulation AFM (FM-AFM), where the frequency of an oscillating cantilever is altered by the gradient of the force that acts between the tip and the sample, has emerged over the years as the method that provides atomic and subatomic spatial resolution as well as force spectroscopy with sub-piconewton sensitivity. FM-AFM is precise; because of all physical observables, time and frequency can be measured by far with the greatest accuracy. By design, FM-AFM clearly separates conservative and dissipative interactions where conservative forces induce a frequency shift and dissipative interactions alter the power needed to maintain a constant oscillation amplitude of the cantilever. As it operates in a noncontact mode, it enables simultaneous AFM and STM measurements. The frequency stability of quartz and the small oscillation amplitudes that are possible with stiff quartz sensors optimize the signal to noise ratio. Here, we discuss the operating principles, the assembly of qPlus sensors, amplifiers, limiting factors, and applications. Applications encompass unprecedented subatomic spatial resolution, the measurement of forces that act in atomic manipulation, imaging and spectroscopy of spin-dependent forces, and atomic resolution of organic molecules, graphite, graphene, and oxides.

Simultaneous AFM Investigation of the Single Cardiomyocyte Electro-Chemo-Mechanics During Excitation-Contraction Coupling.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

Simultaneous AFM and FLIM Imaging with a SiR-DNA Probe Reveals Structural Changes during DNA Condensation in Live Cell Nuclei

We show that the use of fluorescence lifetime imaging (FLIM) on the cell-permeable DNA reporter dye SiR-DNA reports on the condensation state of DNA in the nucleus of live cells. As opposed to other probes, SiR-DNA offers the advantages of minimal toxicity, photostability, and the use of gentler far-red light illumination compared to alternative, UV sensitive probes. This permits single photon excitation and compatibility with super-resolution techniques and makes for a powerful probe for live-cell imaging. Using a variety of biological and chemical processes known to induce DNA condensation and structural modifications, we demonstrate that SiR-DNA FLIM informs on state changes in live nuclei without the conventional requirement for cell fixation with alternative probes. We verify our FLIM findings with simultaneous atomic force microscopy (AFM) measurements in the same field-of-view and monitor nuclear stiffness in response to treatments that affect DNA condensation. The acquisition of such multi-parametric biophysical data enables us to highlight and study differences between types of nuclear modifications and their secondary effects.

Simultaneous AFM and fluorescence imaging: A method for aligning an AFM-tip with an excitation beam using a 2D galvanometer.

Correlative fluorescence and atomic force microscopy (AFM) imaging is a highly attractive technique for use in biological imaging, enabling force and mechanical measurements of particular structures whose locations are known due to the specificity of fluorescence imaging. The ability to perform these two measurements simultaneously (rather than consecutively with post-processing correlation) is highly valuable because it would allow the mechanical properties of a structure to be tracked over time as changes in the sample occur. We present an instrument which allows simultaneous AFM and fluorescence imaging by aligning an incident fluorescence excitation beam with an AFM-tip. Alignment was performed by calibrating a 2D galvanometer present in the excitation beam path and using it to reposition the incident beam. Two programs were developed (one manual and one automated) which correlate sample features between the AFM and fluorescence images, calculating the distance required to translate the incident beam towards the AFM-tip. Using this method, we were able to obtain beam-tip alignment (and therefore field-of-view alignment) from an offset of >15 μm to within one micron in two iterations of the program. With the program running alongside data acquisition for real-time feedback between AFM and optical images, this offset was maintained over a time period of several hours. Not only does this eliminate the need to image large areas with both techniques to ensure that fields-of-view overlap, but it also raises the possibility of using this instrument for tip-enhanced fluorescence applications, a technique in which super-resolution images have previously been achieved.

Insight into diacetylene photopolymerization in Langmuir‐Blodgett films using simultaneous AFM and fluorescence microscopy imaging

A phase‐separated monolayer film comprised of a mixture of 10, 12‐pentacosadiynoic acid (PCDA) with perfluorotetradecanoic acid (PF; CF3(CF2)12COOH) has been characterized using a microscope that is capable of simultaneous atomic force microscope (AFM) imaging and fluorescence imaging. Design criteria for this instrument are described, as well as its application to investigating the mixed film systems. The product of PCDA photopolymerization has two phases, a red phase with strong fluorescence and a blue phase with no fluorescence. With the help of the dual AFM‐fluorescence microscope, it was found that both the red and the blue phase polymers were produced in the same sample from photoillumination, and the relative quantities of the different phases were quantified. Further, the importance of intrinsic mechanical stress in the films is discussed in terms of its influence on selectivity towards the red polymer phase.

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.

Large area fast-AFM scanning with active “Quattro” cantilever arrays

In this work, the fabrication and operation of an active parallel cantilever device integrating four self-sensing and self-actuating probes in an array is presented. The so called “Quattro” cantilever system is controlled by a multichannel field programmable gate array (FPGA) controller. The integrated cantilever devices are fabricated on the basis of a silicon-on-insulator wafer using surface micromachining and gas chopping plasma-etching processes [I. W. Rangelow, J. Vac. Sci. Technol., A 21, 1550 (2003)]. The unique design of the active cantilever probes provides both patterning and readout capabilities [Kaestner et al., J. Micro-Nanolithogr. MEMS 14, 031202 (2015)]. The thermomechanical actuation allows the individually operation of each cantilever in static and dynamic modes. This enables a simultaneous atomic force microscopy operation of all cantilevers in an array, while the piezoresistive read-out of the cantilever bending routinely ensures atomic resolution at a high imaging speed. The scanning probe lithography capabilities of the active cantilevers are based on the utilization of a Fowler–Nordheim field emission process of low-energy electrons (20–50 eV) for direct writing maskless lithography. The cantilever in the Quattro active cantilever array have a pitch of 125 μm (tip-to-tip distance), which allows an image size of 0.5 × 0.2 mm to be acquired within a single scan with 0.2 nm resolution in the vertical direction. Using parallel imaging, an effective scanning speed of 5.6 mm/s is achieved. The multichannel, scalable controller architecture allows four FPGA channels to scan and collect data simultaneously. A data buffer of 128 Mbits for a single frame of 4096 × 1024 pixels is applied. The designed data transfer system allows a packet size of 128 pixels to be transmitted within less than 10 μs, respectively. Thus, the entire image frame is transferred in less than 280 ms, which exceeds the required throughput in the practical cases like critical dimension-metrology and inspection. In this article, the authors are presenting the concept of the system, which combines imaging, metrology, and lithography capabilities with a low-cost of ownership. In this context, the authors are investigating the throughput capability, reproducibility, resolution, and positioning accuracy of the Quattro active cantilever system.

Visualising the strain distribution in suspended two-dimensional materials under local deformation.

We demonstrate the use of combined simultaneous atomic force microscopy (AFM) and laterally resolved Raman spectroscopy to study the strain distribution around highly localised deformations in suspended two-dimensional materials. Using the AFM tip as a nanoindentation probe, we induce localised strain in suspended few-layer graphene, which we adopt as a two-dimensional membrane model system. Concurrently, we visualise the strain distribution under and around the AFM tip in situ using hyperspectral Raman mapping via the strain-dependent frequency shifts of the few-layer graphene’s G and 2D Raman bands. Thereby we show how the contact of the nm-sized scanning probe tip results in a two-dimensional strain field with μm dimensions in the suspended membrane. Our combined AFM/Raman approach thus adds to the critically required instrumental toolbox towards nanoscale strain engineering of two-dimensional materials.

Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks

Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful.