Cell Surface Parameters for Accessing Neutrophil Activation Level with Atomic Force Microscopy.

In this study, we examine the topography and adhesion images of the cell surface of neutrophils during the activation process. Our analysis of cell surface parameters indicates that the most significant changes in neutrophils occur within the first 30 min of activation, suggesting that reactive oxygen species may require approximately this amount of time to activate the cells. Interestingly, we observed surface granular structure as early as 10 min after neutrophil activation when examining atomic force microscopy images. This finding aligns with the reorganization observed within the cells under confocal laser scanning microscopy. By analyzing the cell surface images of adhesion, we identified three spatial surface parameters that correlate with the activation time. This finding enables us to estimate the degree of activation by using atomic force microscopy maps of the cell surface.

Loss of NECTIN1 triggers melanoma dissemination upon local IGF1 depletion.

Cancer genetics has uncovered many tumor-suppressor and oncogenic pathways, but few alterations have revealed mechanisms involved in tumor spreading. Here, we examined the role of the third most significant chromosomal deletion in human melanoma that inactivates the adherens junction gene NECTIN1 in 55% of cases. We found that NECTIN1 loss stimulates melanoma cell migration in vitro and spreading in vivo in both zebrafish and human tumors specifically in response to decreased IGF1 signaling. In human melanoma biopsy specimens, adherens junctions were seen exclusively in areas with low IGF1 levels, but not in NECTIN1-deficient tumors. Our study establishes NECTIN1 as a major determinant of melanoma dissemination and uncovers a genetic control of the response to microenvironmental signals.

Correlation of cell mechanics with the resistance to malignant transformation in naked mole rat fibroblasts.

Naked mole rats (NMRs) demonstrate exceptional longevity and resistance to cancer. Using a biochemical approach, it was previously shown that the treatment of mouse fibroblast cells with RasV12 oncogene and SV40 Large T antigen (viral oncoprotein) led to malignant transformations of cells. In contrast, NMR fibroblasts were resistant to malignant transformations upon this treatment. Here we demonstrate that atomic force microscopy (AFM) can provide information which is in agreement with the above finding, and further, adds unique information about the physical properties of cells that is impossible to obtain by other existing techniques. AFM indentation data were collected from individual cells and subsequently processed through the brush model to obtain information about the mechanics of the cell body (absolute values of the effective Young's moduli). Furthermore, information about the physical properties of the pericellular layer surrounding the cells was obtained. We found a statistically significant decrease in the rigidity of mouse cells after the treatment, whereas there was no significant change found in the rigidity of NMR cells upon the treatment. We also found that the treatment caused a substantial increase in a long part of the pericellular layer in NMR cells only (the long brush was defined as having a size of >10 microns). The mouse cells and smaller brush did not show statistically significant changes upon treatment. The observed change in cell mechanics is in agreement with the frequently observed decrease in cell rigidity during progression towards cancer. The change in the pericellular layer due to the malignant transformation of fibroblast cells has practically not been studied, though it was shown that the removal of part of the pericellular layer of NMR fibroblasts made the cells susceptible to malignant transformation. Although it is plausible to speculate that the observed increase in the long part of the brush layer of NMR cells might help cells to resist malignant transformations, the significance of the observed change in the pericellular layer is yet to be understood. As of now, we can conclude that changes in cell mechanics might be used as an indication of the resistance of NMR cells to malignant transformations.

Can AFM be used to measure absolute values of Young's modulus of nanocomposite materials down to the nanoscale?

At present, a technique potentially capable of measuring values of Young's modulus at the nanoscale is atomic force microscopy (AFM) working in the indentation mode. However, the question if AFM indentation data can be translated into absolute values of the modulus is not well-studied as yet, in particular, for the most interesting case of stiff nanocomposite materials. Here we investigate this question. A special sample of nanocomposite material, shale rock, was used, which is relatively homogeneous at the multi-micron scale. Two AFM modes, force-volume and PeakForce QNM were used in this study. The nanoindentation technique was used as a control benchmark for the measurement of effective Young's modulus of the shale sample. The indentation rate was carefully controlled. To ensure the self-consistency of the mechanical model used to analyze AFM data, the model was modified to take into account the presence of the surface roughness. We found excellent agreement between the average values of effective Young's modulus calculated within AFM and the nanoindenter benchmark method. At the same time, the softest and hardest areas of the sample were seen only with AFM.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

AFM Indentation Analysis of Cells to Study Cell Mechanics and Pericellular Coat.

Atomic force microscopy (AFM) indentation analysis of cells is a unique method of measuring stiffness of the cell body and physical properties of its pericellular coat. These cell parameters correlate with cells of abnormality and diseases. Viable biological cells can be studied with this method directly in a culture dish with no special preparation. Here we describe a step-by-step method to analyze the AFM force-indentation curves to derive cell mechanics (the modulus of elasticity of the cell body) and the parameters of the pericellular coat (density and the thickness of the coat layer). Technical details, potential difficulties, and points of special attention are described.

Imaging of Soft and Biological Samples Using AFM Ringing Mode.

Ringing mode of atomic force microscopy (AFM) enables imaging the surfaces of biological samples, cells, tissue, biopolymers, etc. to obtain unique information, such as the size of molecules pulled by the AFM probe from the sample surface, heights of the sample at different load forces, etc. (up to eight different imaging channels can be recorded simultaneously, which is in addition to five channels already available in other rival modes). The imaging can be done in both air (gases) and liquid (buffers). In addition, the images obtained in ringing mode do not have several common artifacts and can be collected up to 20× faster compared to the rival imaging modes. Here we describe a step-by-step approach to collect images in ringing mode applied to biological and soft materials in general. Technical details, potential difficulties, and points of special attention are described.

Fractal Analysis of Cancer Cell Surface.

Fractal analysis of the cell surface is a rather sensitive method which has been recently introduced to characterize cell progression toward cancer. The surface of fixed and freeze-dried cells is imaged with atomic force microscopy (AFM) modality in ambient conditions. Here we describe the method to perform the fractal analysis specifically developed for the AFM images. Technical details, potential difficulties, points of special attention are described.

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication.

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, <1 × 104 cells ml-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Biophysical differences between chronic myelogenous leukemic quiescent and proliferating stem/progenitor cells.

The treatment of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder has improved recently, but most patients have not yet been cured. Some patients develop resistance to the available tyrosine kinase treatments. Persistence of residual quiescent CML stem cells (LSCs) that later resume proliferation is another common cause of recurrence or relapse of CML. Eradication of quiescent LSCs is a promising approach to prevent recurrence of CML. Here we report on new biophysical differences between quiescent and proliferating CD34+ LSCs, and speculate how this information could be of use to eradicate quiescent LSCs. Using AFM measurements on cells collected from four untreated CML patients, substantial differences are observed between quiescent and proliferating cells in the elastic modulus, pericellular brush length and its grafting density at the single cell level. The higher pericellular brush densities of quiescent LSCs are common for all samples. The significance of these observations is discussed.

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

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.

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 Young's 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 (Young's) 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 Young's 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 Young's modulus between these two types of cells. If we use the model presented here, we see no statistical difference between the values of the Young's 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.

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 Young's 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.

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.

Atomic force microscopy study of nano-physiological response of ladybird beetles to photostimuli.

BACKGROUND: Insects are of interest not only as the most numerous and diverse group of animals but also as highly efficient bio-machines varying greatly in size. They are the main human competitors for crop, can transmit various diseases, etc. However, little study of insects with modern nanotechnology tools has been done. METHODOLOGY/PRINCIPAL FINDINGS: Here we applied an atomic force microscopy (AFM) method to study stimulation of ladybird beetles with light. This method allows for measuring of the internal physiological responses of insects by recording surface oscillations in different parts of the insect at sub-nanometer amplitude level and sub-millisecond time. Specifically, we studied the sensitivity of ladybird beetles to light of different wavelengths. We demonstrated previously unknown blindness of ladybird beetles to emerald color (∼500nm) light, while being able to see UV-blue and green light. Furthermore, we showed how one could study the speed of the beetle adaptation to repetitive flashing light and its relaxation back to the initial stage. CONCLUSIONS: The results show the potential of the method in studying insects. We see this research as a part of what might be a new emerging area of "nanophysiology" of insects.