Cantilever Spring-Constant Calibration in Atomic Force Microscopy

Calibration of AFM cantilever stiffness: a microfabricated array of reflective springs

Force Measurement and Intracellular Operation Using Customized AFM Cantilever

The necessity for development of new drug delivery systems for poorly soluble drugs and therapeutics which targets directly the pathogenic area requires the design of complex systems in nanoscale. Atomic Force Microscopy (AFM) since its discovery offers a unique tool to develop novel drug delivery systems. Its capability to investigate, characterise surfaces and measure forces with spatial resolution at nano-scale respectively contributes to develop and analyse pharmaceutical systems and biomedical devices with complex structures and chemistries. Specifically, polymeric nanoparticles and liposomal drug formulation have been studied extensively by using AFM where their size and morphology where revealed [1]. However, apart from topographical information, AFM can provide details on the local compositions of the sample. There are many modes that can be used to achieve this but the most common is to monitor the phase shift of the oscillating cantilever in tapping mode Such ‘Phase imaging’ can be used to detect nanoscale variation in composition, adhesion, friction, viscoelasticity, and other properties of the materials. Changes in the phase lag often indicate changes in the properties of the sample surface. Phase imaging has proved an extremely useful tool for pharmaceutical characterization. Phase imaging has been used to reveal polymeric forms from single crystal measurements [2] to confirm phase separation of two copolymers for drug delivery [3], to establish the stability of the formulation on different environments [4] and to identify formation of amorphous domains during milling of crystalline salbutamol [5]. Moreover, the ability of AFM to operate on different conditions of temperature humidity and in liquid allows studying the kinetics of range of phenomena. An interesting work was published from Miyazaki et al. where the crystal growth rate of Nifedipine, dispersed in PEG polymer matrix, was quantified [6]. Also, the dissolution process of aspirin on different facets of the crystal was studied by Danesh et al . showing their differences in dissolution rate [7]. The ability of AFM to measure force measurements have been used to investigate interactions among the different compounds in the formulation. To achieve this, the AFM cantilever is modified by attaching a particle directly onto the probe tip and is brought in contact with the substrate of interest, the force required to separate them is the measure of their adhesion. This application of AFM has been used extensively to aerosol drug formulation where the API is associated with a carrier which is released in the site of actions. For instance, AFM has been used to investigate the adhesion force of salbutamol with different excipients [8]. Moreover, localised surface energy and young’s modulus of various compounds have been measured. For example, Young’s modulus were derived from both PLA and PLA/everolimus directly from stent surface on different temperatures. The results showed that everolimus does not affect the mechanical properties of PLA up to 1/1 (w/w) drug loading of the mixture. Also, a significant drop of the Young’s modulus in solution was observed at 36°C, suggests that in vivo the Tg of the polymer is below body temperature [9]. Furthermore, force measurements have been used to identify the encapsulation the efficiency of liposomes as the rigidity of encapsulating liposomes is higher [10].

The interaction force between a rigid silica sphere and a butyl or octyl acetate droplet was measured in an aqueous environment using atomic force microscopy (AFM). The force measurements were performed without added stabilizers and the observed force behavior was found to be dependent on the type of inorganic electrolyte present, where the interfacial tension was constant over the electrolyte concentration range used. Force measurements in the presence of sodium nitrate showed repulsion at all concentrations. Force measurements in the presence of calcium nitrate or sodium perchlorate exhibited an initial repulsion followed by an attraction resulting in a mechanical instability in the AFM cantilever, termed jump-in. The force behavior observed was independent of the water solubility of the organic liquid, in that the same force-distance characteristics were obtained for slightly water soluble butyl acetate and the water sparingly soluble octyl acetate droplets. Modeling of the drop profile during particle-droplet interactions for this type of AFM measurement showed that the force-distance data for the sodium nitrate system obeys typical DLVO interactions. The disagreement between the DLVO predictions for the sodium perchlorate and calcium nitrate systems is attributed to a specific ion effect at the liquid-liquid interface, which gives rise to an attraction force that is greater than the electrostatic double layer repulsion over the length scale of 5 to 10 nm.

In this study we present measurements of the hydrodynamic force exerted on a glass sphere glued to an atomic force microscopy (AFM) cantilever approaching a mica surface in water. A large sphere was used to reduce the impact of the cantilever beam on the measurement. An AFM cantilever with large stiffness was used to accurately determine the actual contact position between the sphere and the sample surface. The measured hydrodynamic force with different approach velocities is in good agreement with the Taylor force calculated in the lubrication theory with the no-slip boundary conditions, which verifies that there is no boundary slip on the glass and mica surfaces. Moreover, a detailed procedure of how to subtract the electrostatic double-layer force is presented.

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

There exists interest in the measurement of small forces for applications such as microtopography of semiconductor devices and atomic force microscopy. A new method is introduced here in which a small silicon beam, that is acted on by the external force of interest, has its position sensed by an rf phase shift technique. The position information in turn is fed back via electrostatic forces to continuously rebalance the beam about its central support. This force-feedback approach provides high sensitivity, submillisecond response, inherent force calibration, and electronically controlled stiffness.

SI-Traceable Spring Constant Calibration of Microfabricated Cantilevers for Small Force Measurement

The measurement of intermolecular forces at the liquid-solid interface is key to many studies of electrochemistry, wetting, catalysis, biochemistry, and mechanobiology. The atomic force microscope (AFM) is unique in its ability to measure and map these forces with nanometer resolution using the oscillating sharp tip of an AFM cantilever. These surface forces are only measured by observing the changes they induce in the dynamics of the resonant AFM probe. However, AFM cantilever dynamics at this interface can be significantly different when compared to air/vacuum environments due to the nature of nanoscale forces at the interface and the low-quality factors in liquids. In this work, we study the nonlinear dynamics of magnetically excited AFM microcantilevers on graphite and mica immersed in deionized water, high-concentration buffers, and methanol. By combining theory and experiments, a wealth of nonlinear dynamical phenomena such as superharmonic resonance, hysteretic jumps, and multimodal interactions are demonstrated and their dependence on hydration/solvation forces is clarified. These results are expected to aid ongoing efforts to link liquid-solid interface properties to cantilever dynamics and lead to accurate interpretation of data from experiments.

After almost 35 years of truly successful and transformative advancements, Atomic Force Microscopy (AFM) and, in general, scanning probe microscopy still have a fundamental limitation. This is constant drift and uncontrolled motion of probe and tested surface structures with respect to each other. This is inherently linked to the currently accepted design principle-only forces are measured, and distances are inferred from force measurements and piezo motions. Here, we demonstrate and test a new setup, which combines advantages of AFM and the surface forces apparatus, where absolute distances are measured by Multiple Beam White Light Interferometry (MBI). The novel and unique aspect of this apparatus consists of a synergistic combination of white light interferometric measurement of the absolute distance by direct reflection from an AFM cantilever and a fast distance clamping and drift correction using an IR-laser Fabry-Pérot interferometry-based approach (FPI). We demonstrate the capabilities of the system by force/distance measurements, benchmarking of distance control by direct comparison of MBI and FPI, and discuss potential applications of the system. This novel setup has the potential to form, monitor, and stress a single molecule or ligand/receptor bond on the molecular hook with sub-nanometer control of molecular distances over in principle infinite times.

Calibration of atomic force microscope (AFM) cantilevers is necessary for the measurementof nanonewton and piconewton forces, which are critical to analytical applications of AFMin the analysis of polymer surfaces, biological structures and organic molecules at nanoscalelateral resolution.We have developed a compact and easy-to-use reference artefact for this calibration, usinga method that allows traceability to the SI (Système International). Traceability is crucial toensure that force measurements by AFM are comparable to those made by optical tweezersand other methods. The new non-contact calibration method measures the springconstant of these artefacts, by a combination of electrical measurements andDoppler velocimetry. The device was fabricated by silicon surface micromachining.The device allows AFM cantilevers to be calibrated quite easily by the ‘cantilever-on-reference’method, with our reference device having a spring constant uncertainty of around ± 5% at one standard deviation. A simple substitution of the analogue velocimeterused in this work with a digital model should reduce this uncertainty to around ± 2%.Both are significant improvements on current practice, and allow traceability to the SI forthe first time at these nanonewton levels.

This paper reports a thermal sensor assembled by two multi-walled carbon nanotubes (MWCNTs) inside a field emission scanning electron microscope (FESEM) which constructed a probe tip in order to detect the local environment of single cell. An atomic force microscope (AFM) cantilever was used for substrate which is composed by Si3N4, and double side was covered by gold layer. Two MWCNTs were assembled to both side of AFM cantilever individually by employing a nanorobotic manipulation. Then another AFM cantilever was used as an end effector to manipulate the MWCNTs to touch, and used electron beam induced deposition (EBID) to bonding the two MWCNTs. The MWCNTs probe thermal sensor was evaluation inside a thermostated container for 30°C to 60°C. The experimental results show the positive characteristics of the temperature coefficient of resistance (TCR). We hope this result will be used rapidly in future. © 2012 IEEE.

Force spectroscopy allows the manipulation of single molecules and the characterization of their properties and interactionsthereby rendering it a powerful tool for biological sciences. Force spectroscopy at the level of individual molecules requires force resolution in the piconewton regime as achieved by optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM) with AFM providing the largest force range from tenth of piconewton to several micronewton. In membrane probe spectroscopy the commonly used sharp cantilever tip is replaced by a lipid-coated glass sphere. This technique expands the scope of force spectroscopy to processes at and between lipid bilayers, like the formation of coiled coils between SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins as well as subsequent membrane fusion. To this end, two solid-supported membranes equipped with SNARE proteins or fusion peptides are separately deposited on a flat glassy surface and on a micrometer glass sphere attached to the end of a tipless AFM cantilever. These two membranes are rapidly brought into contact until a defined force is reached. The AFM deflection readout is used to monitor the distance between the two bilayers, which allows to observe and identify fusion processes of the two lipid membranes, while the forces needed to separate the two surfaces give insights into the formation of SNARE complexes. By changing the contact pressure one can access fusion kinetics and to some extent reconstruct the energy landscape of membrane fusion. In this chapter we describe the preparation of membrane-coated colloidal probes attached to AFM cantilevers, experimental procedures, and necessary data analysis to perform membrane probe spectroscopy in the presence of fusogenic peptides or proteins.

This paper explores strategies for fabricating and maintaining a sharp atomic force microscope (AFM) tip suitable for AFM data storage applications. To this end, AFM cantilevers have been incorporated into micromachined sled carriers and air-bearing sliders. These supports act to limit the maximum loading force on the AFM tip and allow for improved vibration immunity for the AFM cantilever in comparison to macroscopic loading schemes. Readback from a patterned rotating disk has been demonstrated using these devices. Silicon-carbide AFM cantilevers with diamond tips have been fabricated in a process compatible with that of the sleds and sliders. Molded silicon-nitride tips defined by the intersection of three surfaces have also been fabricated for AFM cantilevers. The molds for plane-plane-concave tips are defined by two silicon {111} planes and a silicon-dioxide curved surface. By geometry, the three surfaces necessarily have a unique intersection point, which may improve the consistency in sharpness of these tips relative to conventional pyramidal tips.

The atomic force microscope (AFM) is not only a tool to image the topography of solid surfaces at high resolution. It can also be used to measure force-versus-distance curves. Such curves, briefly called force curves, provide valuable information on local material properties such as elasticity, hardness, Hamaker constant, adhesion and surface charge densities. For this reason the measurement of force curves has become essential in different fields of research such as surface science, materials engineering, and biology. Another application is the analysis of surface forces per se. Some of the most fundamental questions in colloid and surface science can be addressed directly with the AFM: What are the interactions between particles in a liquid? How can a dispersion be stabilized? How do surfaces in general and particles in particular adhere to each other? Particles and surfaces interactions have major implications for friction and lubrication. Force measurements on single molecules involving the rupture of single chemical bonds and the stretching of polymer chains have almost become routine. The structure and properties of confined liquids can be addressed since force measurements provide information on the energy of a confined liquid film. After the review of Cappella [B. Cappella, G. Dietler, Surf. Sci. Rep. 34 (1999) 1–104] 6 years of intense development have occurred. In 1999, the AFM was used only by experts to do force measurements. Now, force curves are used by many AFM researchers to characterize materials and single molecules. The technique and our understanding of surface forces has reached a new level of maturity. In this review we describe the technique of AFM force measurements. Important experimental issues such as the determination of the spring constant and of the tip radius are discussed. Current state of the art in analyzing force curves obtained under different conditions is presented. Possibilities, perspectives but also open questions and limitations are discussed.