Maxim E. Dokukin

Quantitative Mapping of the Elastic Modulus of Soft Materials with HarmoniX and PeakForce QNM AFM Modes

The modulus of elasticity of soft materials on the nanoscale is of interest when studying thin films, nanocomposites, and biomaterials. Two novel modes of atomic force microscopy (AFM) have been introduced recently: HarmoniX and PeakForce QNM. Both modes produce distribution maps of the elastic modulus over the sample surface. Here we investigate the question of how quantitative these maps are when studying soft materials. Three different polymers with a macroscopic Youngs modulus of 0.6-0.7 GPa (polyurethanes) and 2.7 GPa (polystyrene) are analyzed using these new modes. The moduli obtained are compared to the data measured with the other commonly used techniques, dynamic mechanical analyzer (DMA), regular AFM, and nanoindenter. We show that the elastic modulus is overestimated in both the HarmoniX and PeakForce QNM modes when using regular sharp probes because of excessively overstressed material in the samples. We further demonstrate that both AFM modes can work in the linear stress-strain regime when using a relatively dull indentation probe (starting from ~210 nm). The analysis of the elasticity models to be used shows that the JKR model should be used for the samples considered here instead of the DMT model, which is currently implemented in HarmoniX and PeakForce QNM modes. Using the JKR model and ~240 nm AFM probe in the PeakForce QNM mode, we demonstrate that a quantitative mapping of the elastic modulus of polymeric materials is possible. A spatial resolution of ~50 nm and a minimum 2 to 3 nm indentation depth are achieved.

Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments

Here we overview and further develop a quantitative method to measure mechanics of biological cells in indentation experiments, which is based on the use of atomic force microscopy (AFM). We demonstrate how the elastic modulus of the cell body should be measured when the cellular brush is taken into account. The brush is an essential inelastic part of the cell, which surrounds all eukaryotic (the brush is mostly microvilli and glycocalyx) and gram-negative prokaryotic cells (the brush is polysaccharides). The other main feature of the described method is the use of a relatively dull AFM probe to stay in the linear stress-strain regime. In particular, we show that the elastic modulus (aka the Youngs modulus) of cells is independent of the indentation depth up to 10-20% deformation for the eukaryotic cells studied here. Besides the elastic modulus, the method presented allows obtaining the parameters of cellular brush, such as the effective length and grafting density of the brush. Although the method is demonstrated on eukaryotic cells, it is directly applicable for all types of cells, and even non-biological soft materials surrounded by either a brush or any field of long-range forces.

Quantitative Study of the Elastic Modulus of Loosely Attached Cells in AFM Indentation Experiments

When measuring the elastic (Youngs) modulus of cells using AFM, good attachment of cells to a substrate is paramount. However, many cells cannot be firmly attached to many substrates. A loosely attached cell is more compliant under indenting. It may result in artificially low elastic modulus when analyzed with the elasticity models assuming firm attachment. Here we suggest an AFM-based method/model that can be applied to extract the correct Youngs modulus of cells loosely attached to a substrate. The method is verified by using primary breast epithelial cancer cells (MCF-7) at passage 4. At this passage, approximately one-half of cells develop enough adhesion with the substrate to be firmly attached to the substrate. These cells look well spread. The other one-half of cells do not develop sufficient adhesion, and are loosely attached to the substrate. These cells look spherical. When processing the AFM indentation data, a straightforward use of the Hertz model results in a substantial difference of the Youngs modulus between these two types of cells. If we use the model presented here, we see no statistical difference between the values of the Youngs modulus of both poorly attached (round) and firmly attached (close to flat) cells. In addition, the presented model allows obtaining parameters of the brush surrounding the cells. The cellular brush observed is also statistically identical for both types of cells. The method described here can be applied to study mechanics of many other types of cells loosely attached to substrates, e.g., blood cells, some stem cells, cancerous cells, etc.

A biochemical logic approach to biomarker-activated drug release

The present study aims at integrating drug-releasing materials with signal-processing biocomputing systems. Enzymes alanine transaminase (ALT) and aspartate transaminase (AST)—biomarkers for liver injury—were logically processed by a biocatalytic cascade realizing a Boolean AND gate. Citrate produced in the system was used to trigger a drug-mimicking release from alginate microspheres. In order to differentiate low vs. high concentration signals, the microspheres were coated with a protective shell composed of layer-by-layer adsorbed poly(L-lysine) and alginate. The alginate core of the microspheres was prepared from Fe3+-cross-linked alginate loaded with rhodamine 6G dye mimicking a drug. Dye release from the core occurred only when both biomarkers, ALT and AST, appeared at their high pathophysiological concentrations jointly indicative of liver injury. The signal-triggered response was studied at the level of a single microsphere, yielding information on the dye release kinetics.

Emergence of fractal geometry on the surface of human cervical epithelial cells during progression towards cancer

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.

Detection of cancerous cervical cells using physical adhesion of fluorescent silica particles and centripetal force.

Here we describe a non-traditional method to identify cancerous human cervical epithelial cells in a culture dish based on physical adhesion between silica beads and cells. It is a simple optical fluorescence-based technique which detects the relative difference in the amount of fluorescent silica beads physically adherent to surfaces of cancerous and normal cervical cells. The method utilizes the centripetal force gradient that occurs in a rotating culture dish. Due to the variation in the balance between adhesion and centripetal forces, cancerous and normal cells demonstrate clearly distinctive distributions of the fluorescent particles adherent to the cell surface over the culture dish. The method demonstrates higher adhesion of silica particles to normal cells compared to cancerous cells. The difference in adhesion was initially observed by atomic force microscopy (AFM). The AFM data were used to design the parameters of the rotational dish experiment. The optical method that we describe is much faster and technically simpler than AFM. This work provides proof of the concept that physical interactions can be used to accurately discriminate normal and cancer cells.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

UNLABELLED We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

On averaging force curves over heterogeneous surfaces in atomic force microscopy.

Atomic force microscopy (AFM) can be used to study mechanics at the nanoscale. Biological surfaces and nanocomposites have typically heterogeneous surfaces, both mechanically and chemically. When studying such surfaces with AFM, one needs to collect a large amount of data to make statistically sound conclusions. It is time- and resource-consuming to process each force curve separately. The analysis of an averaged raw force data is a simple and time saving option, which also averages out the noise and measurement artifacts of the force curves being analyzed. Moreover, some biomedical applications require just an average number per biological cell. Here we investigate such averaging, study the possible artifacts due to the averaging, and demonstrate how to minimize or even to avoid them. We analyze two ways of doing the averaging: over the force data for each particular distance (method 1, the most commonly used way), and over the distances for each particular force (method 2). We derive the errors of the methods in finding to the true average rigidity modulus. We show that both methods are accurate (the error is <2%) when the heterogeneity of the surface rigidity is small (<50%). When the heterogeneity is large (>100×), method 2 underestimates the average rigidity modulus by a factor of 2, whereas the error of method 1 is only 15%. However, when analyzing the different surface chemistry, which reveals itself in the changing long-range forces, the accuracy of the methods behave oppositely: method 1 can produce a noticeable averaging artifact in the deriving of the long-range forces; whereas method 2 can be successfully used to derive the averaged long-range force parameters without artifacts. We exemplify our conclusions by the study of human cervical cancer and normal epithelial cells, which demonstrate different degrees of heterogeneity.

High-resolution high-speed dynamic mechanical spectroscopy of cells and other soft materials with the help of atomic force microscopy

Dynamic mechanical spectroscopy (DMS), which allows measuring frequency-dependent viscoelastic properties, is important to study soft materials, tissues, biomaterials, polymers. However, the existing DMS techniques (nanoindentation) have limited resolution when used on soft materials, preventing them from being used to study mechanics at the nanoscale. The nanoindenters are not capable of measuring cells, nanointerfaces of composite materials. Here we present a highly accurate DMS modality, which is a combination of three different methods: quantitative nanoindentation (nanoDMA), gentle force and fast response of atomic force microscopy (AFM), and Fourier transform (FT) spectroscopy. This new spectroscopy (which we suggest to call FT-nanoDMA) is fast and sensitive enough to allow DMS imaging of nanointerfaces, single cells, while attaining about 100x improvements on polymers in both spatial (to 10–70 nm) and temporal resolution (to 0.7s/pixel) compared to the current art. Multiple frequencies are measured simultaneously. The use of 10 frequencies are demonstrated here (up to 300 Hz which is a rather relevant range for biological materials and polymers, in both ambient conditions and liquid). The method is quantitatively verified on known polymers and demonstrated on cells and polymers blends. Analysis shows that FT-nanoDMA is highly quantitative. The FT-nanoDMA spectroscopy can easily be implemented in the existing AFMs.

Atomic force microscopy to detect internal live processes in insects

Here we report on the use of atomic force microscopy (AFM) to study surface oscillations coming from internal live processes of insects. With a specially designed AFM stage to keep an insect motion partially restricted, the AFM can record internal oscillations on different parts of the insect. We demonstrate the method for a fly, mosquito, and lady beetle. We show that AFM can provide information about the spectral behavior that has not been studied so far, 10–600 Hz range, detecting amplitudes down to subnanometer level.