Atomic Force Microscopy Force Mapping Research Articles

A method for analyzing AFM force mapping data obtained from soft tissue cryosections

Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue’s highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.

Local intracellular stiffening of ascidian embryo in cleavage developmental stage observed by atomic force microscopy

During the cleavage stage in ascidian embryos, cells undergo repeated and regular cell division in the animal hemisphere. We investigated how the intracellular stiffness, that is, the apparent Young’s modulus of cells in the animal hemisphere changes in the cleavage stages from the 16-cell to 76-cell stages using atomic force microscopy (AFM). The AFM force mapping experiments showed that a pair of dot-like stiffening clearly appeared in the intracellular region, which involves the cortical and cytoplasmic regions indented by the AFM probe, in a period of the interphase after cell division. The dot-like structures disappeared before beginning to increase the whole cell stiffening towards the proceeding mitotic phase. We found that the positioning of a pair of dots was located so as to intersect the daughter cells formed in the proceeding cell division. These results suggest that the local intracellular stiffening observed in the interphase may play an important role in controlling the orientation of the mitotic spindle that determines the direction of cell division.

Mechanical characterization of electrospun polyesteretherurethane (PEEU) meshes by atomic force microscopy

The mechanical properties of electrospun fiber meshes typically are measured by tensile testing at the macro-scale without precisely addressing the spatial scale of living cells and their submicron architecture. Atomic force microscopy (AFM) enables the examination of the nano- and micro-mechanical properties of the fibers with potential to correlate the structural mechanical properties across length scales with composition and functional behavior. In this study, a polyesteretherurethane (PEEU) polymer containing poly(p-dioxanone) (PPDO) and poly(ɛ-caprolactone) (PCL) segments was electrospun into fiber meshes or suspended single fibers. We employed AFM three point bending testing and AFM force mapping to measure the elastic modulus and stiffness of individual micro/nanofibers and the fiber mesh. The local stiffness of the fiber mesh including the randomized, intersecting structure was also examined for each individual fiber. Force mapping results with a set point of 50 nN demonstrated the dependence of the elasticity of a single fiber on the fiber mesh architecture. The non-homogeneous stiffness along the same fiber was attributed to the intersecting structure of the supporting mesh morphology. The same fiber measured at a point with and without axial fiber support showed a remarkable difference in stiffness, ranging from 0.2 to 10 nN/nm respectively. For the region, where supporting fibers densely intersected, the stiffness was found to be considerably higher. In the region where the degrees of freedom of the fibers was not restricted, allowing greater displacement, the stiffness were observed to be lower. This study elucidates the relationship between architecture and the mechanical properties of a micro/nanofiber mesh. By providing a greater understanding of the role of spatial arrangement and organization on the surface mechanical properties of such materials, we hope to provide insight into the design of microenvironments capable of regulating cell functionality.

Variations of Elastic Modulus and Cell Volume with Temperature for Cortical Neurons

Neurons change their growth dynamics and mechanical properties in response to external stimuli such as stiffness of the local microenvironment, ambient temperature, and biochemical or geometrical guidance cues. Here we use combined atomic force microscopy (AFM) and fluorescence microscopy experiments to investigate the relationship between external temperature, soma volume, and elastic modulus for cortical neurons. We measure how changes in ambient temperature affect the volume and the mechanical properties of neuronal cells at both the bulk (elastic modulus) and local (elasticity maps) levels. The experimental data demonstrate that both the volume and the elastic modulus of the neuron soma vary with changes in temperature. Our results show a decrease by a factor of 2 in the soma elastic modulus as the ambient temperature increases from room (25 °C) to physiological (37 °C) temperature, while the volume of the soma increases by a factor of 1.3 during the same temperature sweep. Using high-resolution AFM force mapping, we measure the temperature-induced variations within different regions of the elasticity maps (low and high values of elastic modulus) and correlate these variations with the dynamics of cytoskeleton components and molecular motors. We quantify the change in soma volume with temperature and propose a simple theoretical model that relates this change with variations in soma elastic modulus. These results have significant implications for understanding neuronal development and functions, as ambient temperature, cytoskeletal dynamics, and cellular volume may change with variations in physiological conditions, for example, during tissue compression and infections in vivo as well as during cell manipulation and tissue regeneration ex vivo.

Topography effects in AFM force mapping experiments on xylan-decorated cellulose thin films

Abstract Xylan-coated cellulose thin films has been investigated by means of atomic force microscopy (AFM) and force mapping experiments. The birch xylan deposition on the film was performed under control by means of a multiple parameter surface plasmon resonance spectroscopy (MP-SPR) under dynamic conditions. The coated films were submitted to AFM in phase imaging mode to force mapping with modified AFM tips (sensitive to hydrophilic OH and hydrophobic CH3 groups) in order to characterize and localize the xylan on the surfaces. At the first glance, a clear difference in the adhesion force between xylan-coated areas and cellulose has been observed. However, these different adhesion forces originate from topography effects, which prevent an unambiguous identification and subsequent localization of the xylan on the cellulosic surfaces.

Could Atomic-Force Microscopy Force Mapping Be a Fast Alternative to Core-Plug Tests for Optimizing Injection-Water Salinity for Enhanced Oil Recovery in Sandstone?

Summary Core tests demonstrated that decreasing the salinity of injection water can increase oil recovery. Optimizing injection-water salinity, however, would offer a clear economic advantage for several reasons. Too-low salinity risks swelling of the clays that would lead to permanent reservoir damage, but evidence of effectiveness with moderate-salinity solutions would make it less difficult to dispose of produced water. The goal is to define boundary conditions so injection-water salinity is high enough to prevent reservoir damage and low enough to induce the low-salinity (LS) effect, while keeping costs and operational requirements at a minimum. Traditional core-plug testing for optimizing conditions has some limitations. Each test requires a fresh sample; core-testing requires sophisticated and expensive equipment; and reliable core-test data require several months because cores must be cleaned, restored, and aged before the tests can begin. It is also difficult to compare data from one core with results from another because no two cores are identical, making it difficult to distinguish between effects resulting from different conditions and effects resulting from different cores. Gathering statistics is limited by the time required for each test and the fact that core material is in short supply. Thus, our aim was to explore the possibility of a less-expensive, faster alternative by probing the fundamental chemical mechanisms behind the LS effect. We developed a method that uses atomic-force microscopy (AFM) to investigate the relationship between the wettability of pore surfaces and water salinity. We functionalize AFM tips with organic molecules and use them to represent tiny oil droplets of nonpolar molecules, and we use sand grains removed from core plugs to represent the pore walls in sandstone. We bring our “oil”-wet tip close to the sand-grain surface and measure the work of adhesion between the tip and the surface. Repeated probing of the surface with the tip produces data that one can convert to maps of adhesion, and we can estimate contact angle. Adhesion work is proportional to wettability and is directly correlated with the salinity of the fluid in contact with the tip and the particle surface. From our measurements, the threshold values for the onset of the LS response are 5,000 to 8,000 ppm, which benchmark remarkably well with observations from core-plug tests. From a mechanistic perspective, the correlation between salinity and adhesion provides evidence for the role of electrical-double-layer (EDL) expansion in the LS response; expansion of the double layer decreases oil wettability. Because AFM experiments can be performed relatively quickly on very little material, they give the possibility of testing salinity response on many samples throughout a reservoir and for gathering statistics. Our approach provides a range of data that one can use to screen conditions to maximize the value of the core-plug testing and to provide extra data that would be too time consuming or too expensive to gather with traditional methods alone. Thus, AFM force mapping is an excellent complement to traditional core-plug testing.

Determination of local and global elastic moduli of valve interstitial cells cultured on soft substrates

The elasticity of the extracellular matrix profoundly affects biological responses of cells, but also their mechanical properties. Single cell mechanical properties are often measured by atomic force microscopy (AFM), but technical guidelines for AFM measurement of cells grown on soft substrates are not well established. In this study, the local and global elastic moduli of aortic valve interstitial cells (VICs) cultured on soft polyacrylamide substrates (3–144kPa) were characterized via AFM force mapping using pyramidal and spherical tips, respectively. Local and global VIC modulus values both increased with substrate stiffness (p<0.05), with the average local cell modulus being consistently two to three times greater than the global modulus (p<0.05). For local measurements, a minimum of four measurements was required to observe the trend of increasing cell modulus with substrate stiffness, but there was no advantage to testing additional spots. The Hertz model was relatively accurate in estimating the global cell elastic modulus (<12% error, based on validated finite element analyses), despite the cells being of finite thickness and grown on deformable substrates, neither of which are accounted for in the Hertz model. The results of this study provide practical guidelines for efficient AFM-based measurement of the mechanical properties of cells on gels. They also provide new physiologically-relevant data on VIC mechanical properties and their correlation with substrate stiffness-dependent cytoskeletal changes, with relevance to heart valve mechanobiology and disease.

Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (β-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, β-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.

Evaluation of Lamina Cribrosa and Peripapillary Sclera Stiffness in Pseudoexfoliation and Normal Eyes by Atomic Force Microscopy

Pseudoexfoliation (PEX) syndrome is a systemic disorder of the elastic fiber system that can lead to PEX glaucoma. Elastotic alterations in the lamina cribrosa (LC) of PEX eyes suggested biomechanical implications predisposing to pressure-induced optic nerve damage. In this pilot study, the stiffness of LC and peripapillary sclera (ppSC) in eyes with and without PEX syndrome were analyzed by atomic force microscopy (AFM) nanoindentation. Unfixed cryosections (5-μm thick) were prepared from the optic nerve heads (ONH) of three donor eyes with PEX syndrome and three age-matched control eyes. AFM force mapping was performed in selected regions of the central, midperipheral, and peripheral LC and the ppSC using a spherical cantilever tip. To determine the local Young's modulus of elasticity (YME) as a measure of tissue stiffness, force curves were acquired and analyzed using the spherical Hertz model. For the LC, the median YME values calculated from single stiffness maps averaged 17.2 (±2.7) kPa in normal eyes and 10.1 (±1.4) kPa in PEX eyes, indicating a significant PEX-related decrease in stiffness by over 40% (P < 0.01). The corresponding YME values for the ppSC, which revealed a 9-fold higher tissue stiffness than in the LC, averaged 158.3 (±59.8) kPa for control and 85.8 (±16.9) kPa for PEX samples. AFM was proven suitable for determining the stiffness of ONH tissues, encouraging further large-scale analyses. The marked decrease in stiffness, implying an increased deformability of the ONH in PEX eyes, may reflect an inherent tissue weakness rendering these eyes more vulnerable to glaucomatous damage.

CsgA Production by Escherichia coli O157:H7 Alters Attachment to Abiotic Surfaces in Some Growth Environments

The role of curli expression in attachment of Escherichia coli O157:H7 to glass, Teflon, and stainless steel (SS) was investigated through the creation of csgA knockout mutants in two isolates of E. coli O157:H7. Attachment assays using epifluorescence microscopy and measurements of the force of adhesion of bacterial cells to the substrates using atomic force microscopy (AFM) force mapping were used to determine differences in attachment between wild-type (wt) and csgA-negative (ΔcsgA) strains following growth in four different media. The hydrophobicity of the cells was determined using contact angle measurements (CAM) and bacterial adhesion to hydrocarbons (BATH). The attachment assay results indicated that ΔcsgA strains attached to glass, Teflon, and SS surfaces in significantly different numbers than their wt counterparts in a growth medium-dependent fashion (P < 0.05). However, no clear correlation was seen between attachment numbers, surface type, or growth medium. No correlation was seen between BATH and CAM results (R(2) < 0.70). Hydrophobicity differed between the wt and ΔcsgA in some cases in a growth medium- and method-dependent fashion (P < 0.05). AFM force mapping revealed no significant difference in the forces of adhesion to glass and SS surfaces between wt and ΔcsgA strains (P > 0.05) but a significantly greater force of adhesion to Teflon for one of the two wt strains than for its ΔcsgA counterpart (P < 0.05). This study shows that CsgA production by E. coli O157:H7 may alter attachment behavior in some environments; however, further investigation is required in order to determine the exact relationship between CsgA production and attachment to abiotic surfaces.

Abstract 416: Mammographic density and ECM stiffness

Abstract Breast tissue homoeostasis depends on the complex regulation of biochemical and biophysical factors present in the extra cellular matrix (ECM). Tumor tissue in mouse and human breast is characteristically stiffer than normal tissue, and changes to the ECM contribute to this phenotype. Breast density is associated with elevated collagen content, while increased deposition of collagen is known to augment ECM stiffness. Mammographic density imaging provides a qualitative description of breast density, and high mammographic density is associated with increased risk to malignancy. However, little is known about the correlation between ECM stiffness and the density in the breast image. Previously, we have used atomic force microscopy (AFM) to characterize the changes in breast tissue stiffness with cancer progression (Levental et al., Cell 2009). We hypothesized that mammographically dense breasts would be stiffer. To further understand the biophysical regulation of breast stiffness, we investigated whether mammographic density is a predictor of ECM stiffness. Towards this goal, we studied tissue samples from prophylactic mastectomies, from women with low versus high mammographic density. Tissue samples were obtained from the peripheral quadrants as well as from the retroareolar (nipple) regions, which are known to be of higher mammographic density. We performed AFM force mapping to characterize the ECM stiffness in the different regions. Our preliminary data indicate a heterogeneous pattern of stiffness within the breast. Moreover, we found that the peripheral terminal ductal lobuli have the most compliant ECM. By contrast, the stroma stiffness in the retroareolar region was 2-fold higher than in the peripheral quadrants. This work demonstrates the technical feasibility of measuring the mechanical properties as well as defined histological characteristics within the same patient specimen. The data suggest that ECM stiffness may be greater in breast tissue of high mammographic density. The correlation between mammographic density and ECM stiffness may reveal the role of collagen status in the risk to malignancy. (supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2011-416

AFM Force Mapping for Characterizing Patterns of Electrostatic Charges on SiO2 Electrets

To characterize patterns of charges on electrets, Kelvin probe force microscopy (KFM) usually serves as a very useful tool to measure the electrostatic potential through an electric cycle; however, it is limited to electrets supported on conductive substrates. In this article, we demonstrate the use of atomic force microscopy (AFM) force mapping to visualize the pattern of charges on SiO(2) electrets. In contrast to KFM, AFM force mapping can be used for characterizing electrets that are formed not only on conductive substrates but also on nonconductive substrates. Functional groups can be introduced to the AFM tip to achieve the force mapping and to improve the resolution. Our study clearly indicates that AFM force mapping can serve as an optional method for the characterization of electrets.

Phase Segregation of Untethered Zwitterionic Model Lipid Bilayers Observed on Mercaptoundecanoic-Acid-Modified Gold by AFM Imaging and Force Mapping

Planar supported lipid bilayers (SLBs) are often studied as model cell membranes because they are accessible to a variety of surface-analytic techniques. Specifically, recent studies of lipid phase coexistence in model systems suggest that membrane lateral organization is important to a range of cellular functions and diseases. We report the formation of phase-segregated dioleoylphosphatidylcholine (DOPC)/sphingomyelin/cholesterol bilayers on mercaptoundecanoic-acid-modified (111) gold by spontaneous fusion of unilamellar vesicles, without the use of charged or chemically modified headgroups. The liquid-ordered (l(o)) and liquid-disordered (l(d)) domains are observed by atomic force microscopy (AFM) height and phase imaging. Furthermore, the mechanical properties of the bilayer were characterized by force-indentation maps. Fits of force indentation to Sneddon mechanics yields average apparent Young's moduli of the l(o) and l(d) phases of 100 +/- 2 and 59.8 +/- 0.9 MPa, respectively. The results were compared to the same lipid membrane system formed on mica with good agreement, though modulus values on mica appeared higher. Semiquantitative comparisons suggest that the mechanical properties of the l(o) phase are dominated by intermolecular van der Waals forces, while those of the fluid l(d) phase, with relatively weak van der Waals forces, are influenced appreciably by differences in surface charge density between the two substrates, which manifests as a difference in apparent Poisson ratios.

Nanomechanical Properties of Supramolecular Self-Assembled Whiskers Determined by AFM Force Mapping

In this Letter, we investigate the nanomechanical properties of self-assembled 1,3,5-benzenetrisamide whiskers with atomic force microscopy (AFM) bending experiments. We use force mapping to acquire spatially resolved force measurements over the full length of a whisker segment spanning a channel of a structured glass substrate. This allows validation of the experimental boundary conditions directly from the AFM data and a reliable determination of Young's modulus. The presented technique can be generalized for the mechanical characterization of other one-dimensional materials.

Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: Atomic force microscopy force mapping of pulmonary endothelial monolayer

Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.

PRECISION MAPPING OF ERYTHROCYTE TRAUMA BY ATOMIC FORCE MICROSCOPY

Over the past years experimental advances have made it possible to elucidate the components of the cell membrane and its structure, thus rendering the measurement of micro-mechanical properties much more efficient than previously. This study investigates the use of Atomic Force Microscopy (AFM) to discern subtle alterations to the red cell due to exposure within artificial organs. Red cells were stressed both biochemically and by controlled exposure to supra-physiological shear. AFM imaging was performed using a Multimode AFM (Nanoscope IIIA, Digital Instruments), using tapping mode, contact mode, and force-volume imaging. Force maps obtained using force-volume imaging were analysed using Igor Pro software (Wavemetrics), and macros developed in our laboratory (by N. Almqvist). Preliminary force maps have been obtained on specimens of human erythrocytes: both in a stress-free condition and in a tensed or echinocytic condition. Ongoing finite-element simulation models are being developed to compliment these experimental studies, with the ultimate goal of inverting nano-scale elastic properties of the blood cells. This preliminary success encourages the use of AFM force mapping as a sensitive tool to evaluate sub-lytic trauma to red blood cells. Future studies aim to investigate the elasticity and adhesion other relevant cells including leukocytes and platelets.