Atomic Force Microscopy Calibration Research Articles

Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Is It Possible to Directly Determine the Radius of a Spherical Indenter Using Force Indentation Data on Soft Samples?

An important factor affecting the accuracy of Young's modulus calculation in Atomic Force Microscopy (AFM) indentation experiments is the determination of the dimensions of the indenter. This procedure is usually performed using AFM calibration gratings or Scanning Electron Microscopy (SEM) imaging. However, the aforementioned procedure is frequently omitted because it requires additional equipment. In this paper, a new approach is presented that focused on the calibration of spherical indenters without the need of special equipment but instead using force indentation data on soft samples. Firstly, the question whether it is mathematically possible to simultaneously calculate the indenter's radius and the Young's modulus of the tested sample (under the restriction that the sample presents a linear elastic response) using the same force indentation data is discussed. Using a simple mathematical approach, it was proved that the aforementioned procedure is theoretically valid. In addition, to test this method in real indentation experiments agarose gels were used. Multiple measurements on different agarose gels showed that the calibration of a spherical indenter is possible and can be accurately performed. Thus, the indenter's radius and the soft sample's Young's modulus can be determined using the same force indentation data. It is also important to note that the provided accuracy is similar to the accuracy obtained when using AFM calibration gratings. The major advantage of this paper is that it provides a method for the simultaneous determination of the indenter's radius and the sample's Young's modulus without requiring any additional equipment.

A novel PRC signal drift reduction method for new developed SEM-based nanoindentation/nanoscratch instrument integrated with AFM

In-situ SEM nanoindentation and nanoscratch testing methods are commonly used for mechanical characterization and investigation of the deformation and failure mechanisms of coating materials with micro-to nano-scale thicknesses. However, existing SEM-based integrated nanoindentation and nanoscratch instruments have two main limitations. First, the measured mechanical properties of the coating materials at micro-to nano-scale thicknesses are highly sensitive to surface roughness. Second, the existing SEM-based instruments lack the capability to acquire the morphology of residual imprints in real-time after nanoindentation and nanoscratching. In this study, a novel SEM-based integrated nanoindentation, nanoscratch, and atomic force microscopy (AFM) instrument, namely, NMT-AFM was proposed, developed and fabricated. The self-sensing piezoresistive cantilever (PRC) was selected as the AFM force sensor owing to its miniaturization ability. However, the resistance of the PRC sensor fluctuated because of the electron irradiation from SEM, resulting in the continuous drift of the PRC signal during SEM imaging. To overcome this limitation, a mechanism of PRC signal drift inside SEM was analyzed for the first time, and a PRC signal drift reduction method was proposed based on the mechanism analysis. The experimental results indicated that the PRC signal drift was reduced to 2 nm in 2 min by applied external voltage value UA of 30 V to modified PRC, which proved the proposed mechanism of PRC signal drift during SEM imaging. Finally, the X–Y fine nanopositioner angle calibration test using AFM calibration chip VGRP-UM and the nanoindentation/nanoscratch characterizations of the TiAlSiN coating material were conducted.

Study of low energy ion beam induced sputtering parameters of muscovite mica [KAl2(Si3Al)O10(OH)2] using Monte Carlo simulation

Naturally available Muscovite mica [KAl2(Si3Al)O10(OH)2] is an multi-elemental insulating 2D material for widely used in atomic force microscopy calibration, biological substrate deposition, electronic device material etc. The wide application and easy availability of mica with very low cost can be used to tailor different properties by modifying several or mono layer using sputtering of different constituent elements of mica with varying ion beam parameters. Since the ion beam technique is a versatile, precise, and controlled way, the modification of single or multiple layers is possible by choosing proper parameters. The modification of several layers of mica surface is basically the consequence of sputtering of different mica elements, which can be understood by the sputtering yield (Y) of mica elements. The trend of sputtering yield with ion incidence angle, projectile mass, and ion energy is studied here through Monte Carlo Simulation using Stopping and Range of Ions in Matter (SRIM) freeware. The sputtering yield of potassium (K) atoms as well as mica is found to increase first with ion incidence angle and becomes maximum around 70° and 80° for 500 eV and 10 keV ion, respectively; then decreases for all the ions considered. The potassium (K) vacancy profile is calculated and found to be consistent with sputtering yield results. The sputtering yield of mica increases with increase of projectile ion mass and ion energy except for He+ ion, which is explained using ion penetration depth.

One-Step Calibration of AFM in Liquid

Nanomechanical measurements of cells and single molecules with atomic force microscopy (AFM) require accurate calibration of two parameters: the spring constant of the cantilever (k) and the inverse of the optical lever sensitivity (InvOLS). The most established calibration approach in liquid consists in determining the InvOLS from force–distance curves on a stiff surface, k being calculated using the thermal spectrum (PSD) of the cantilever via the equipartition theorem. Recent works proposed using cantilevers with calibrated k and then determining the InvOLS from the PSD. These non-contact approaches improve the precision of nanomechanical measurements compared to conventional contact-based approaches. The Sader method or the recent global calibration initiative (GCI) are accurate approaches and do not require knowledge of the InvOLS to determine k, thus they would allow one-step calibration of AFM in liquid. However, both methods assume high quality factor cantilevers, not the case for most cantilevers in liquid. Here we assess the accuracy and precision of the Sader and GCI methods in liquid on two types of cantilevers with low Q-factor using two different PSD fitting models (SHO and Pirzer). We evaluate the two approaches using only the PSD in liquid to calibrate both k and the InvOLS. While both methods led to similar results, the GCI approach is less prone to systematic uncertainties and, using the SHO model, provides higher accuracy in k and the InvOLS. Therefore, the proposed SHO, GCI-based approach utilizing only the thermal spectrum in liquid is precise and accurate and allows one-step calibration of AFM.

Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy.

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

Elastic moduli of cellulose nanofibers isolated from various cellulose resources by using aqueous counter collision

An atomic force microscopy (AFM) three-points bending (TPB) test is very useful to measure the mechanical properties of cellulose nanofibers (CNFs). This study investigates the Young’s moduli of various CNFs by using AFM TPB test. CNFs were isolated from hardwood, softwood, bamboo and cotton by using an aqueous counter collision (ACC) method. Single CNF was transferred to the AFM calibration chip by using contact transfer for the AFM TPB test and the CNF was considered as a clamped-clamped beam. An Euler equation was used to calculate Young’s modulus under linear deformation assumption. The cross section of single CNF is assumed to be circular shape. Young’s moduli of hardwood, softwood, bamboo and cotton CNFs isolated by ACC method are in the range of 88–110 GPa, which are comparable with the previously reported values.

A sub-50 nm three-step height sample for AFM calibration

In this paper, a sub-50 nm three-step height sample was made for vertical calibration of atomic force microscopy (AFM) and a new step height evaluation algorithm based on polynomial fitting is discussed. The influences of AFM artefacts such as particles, image bow and high-order errors on step height were studied. The experimental results showed that the polynomial order p2 and threshold t were not critical factors. However, the increment Δh and the polynomial order p used in the calculation of optimal shifting distance were important and must be carefully considered. Δh = 0.1 nm and p ≥ 4 were determined to get a stable step height. The sample had small roughness and good uniformity. It has the potential to serve as a high quality step height standard sample for AFM calibration.

Corrected direct force balance method for atomic force microscopy lateral force calibration

This paper reports corrections and improvements of the previously reported direct force balance method (DFBM) developed for lateral calibration of atomic force microscopy. The DFBM method employs the lateral force signal obtained during a force-distance measurement on a sloped surface and relates this signal to the applied load and the slope of the surface to determine the lateral calibration factor. In the original publication [Rev. Sci. Instrum. 77, 043903 (2006)], the tip-substrate contact was assumed to be pinned at the point of contact, i.e., no slip along the slope. In control experiments, the tip was found to slide along the slope during force-distance curve measurement. This paper presents the correct force balance for lateral force calibration.

Spring constant calibration of atomic force microscopy cantilevers with a piezosensor transfer standard

We describe a method to calibrate the spring constants of cantilevers for atomic force microscopy (AFM). The method makes use of a "piezosensor" composed of a piezoresistive cantilever and accompanying electronics. The piezosensor was calibrated before use with an absolute force standard, the NIST electrostatic force balance (EFB). In this way, the piezosensor acts as a force transfer standard traceable to the International System of Units. Seven single-crystal silicon cantilevers with rectangular geometries and nominal spring constants from 0.2 to 40 Nm were measured with the piezosensor method. The values obtained for the spring constant were compared to measurements by four other techniques: the thermal noise method, the Sader method, force loading by a calibrated nanoindentation load cell, and direct calibration by force loading with the EFB. Results from different methods for the same cantilever were generally in agreement, but differed by up to 300% from nominal values. When used properly, the piezosensor approach provides spring-constant values that are accurate to +/-10% or better. Methods such as this will improve the ability to extract quantitative information from AFM methods.

Standardized procedure for calibrating height scales in atomic force microscopy on the order of 1 nm

We propose a standard stepped sample and specify a procedure for determining the step height for height-scale calibration of atomic force microscopy (AFM) instruments on the order of sub-nm to nm. The stepped Si(111) surface structure was confirmed to be the most appropriate for the standard sample by a series of round-robin measurements carried out by a group comprising AFM instrument manufacturers and user organizations. By specifying a standard measurement procedure, the mean standard deviation of the step heights is decreased by 50%. It is also confirmed that the measured Si step height is accurate to ±5%, which is consistent with the accuracy obtained for higher step standard samples. A prototype common data processing software program containing the algorithm of the specified procedure was used to allow direct comparison of the untreated measurement data from each organization.