Reducing uncertainties in energy dissipation measurements in atomic force spectroscopy of molecular networks and cell-adhesion studies
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Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) is a valuable tool in biophysics to investigate the ligand-receptor interactions, cell adhesion and cell mechanics. However, the force spectroscopy data analysis needs to be done carefully to extract the required quantitative parameters correctly. Especially the large number of molecules, commonly involved in complex networks formation; leads to very complicated force spectroscopy curves. One therefore, generally characterizes the total dissipated energy over a whole pulling cycle, as it is difficult to decompose the complex force curves into individual single molecule events. However, calculating the energy dissipation directly from the transformed force spectroscopy curves can lead to a significant over-estimation of the dissipated energy during a pulling experiment. The over-estimation of dissipated energy arises from the finite stiffness of the cantilever used for AFM based SMFS. Although this error can be significant, it is generally not compensated for. This can lead to significant misinterpretation of the energy dissipation (up to the order of 30%). In this paper, we show how in complex SMFS the excess dissipated energy caused by the stiffness of the cantilever can be identified and corrected using a high throughput algorithm. This algorithm is then applied to experimental results from molecular networks and cell-adhesion measurements to quantify the improvement in the estimation of the total energy dissipation.Keywords:
Force Spectroscopy
Force Spectroscopy
Multicellular organism
Cell–cell interaction
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Macroscopic assays that are traditionally used to investigate the adhesion behaviour of microbial cells provide averaged information obtained on large populations of cells and do not measure the fundamental forces driving single-cell adhesion. Here, we use single-cell force spectroscopy (SCFS) to quantify the specific and non-specific forces engaged in the adhesion of the human fungal pathogen Candida albicans. Saccharomyces cerevisiae cells expressing the C. albicans adhesion protein Als5p were attached on atomic force microscope tipless cantilevers using a bioinspired polydopamine wet polymer, and force–distance curves were recorded between the obtained cell probes and various solid surfaces. Force signatures obtained on hydrophobic substrates exhibited large adhesion forces (1.25 ± 0.2 nN) with extended rupture lengths (up to 400 nm), attributed to the binding and stretching of the hydrophobic tandem repeats of Als5p. Data collected on fibronectin (Fn)-coated substrates featured strong adhesion forces (2.8 ± 0.6 nN), reflecting specific binding between Fn and the N-terminal immunoglobulin-like region of Als5p, followed by weakly adhesive macromolecular bonds. Both hydrophobic and Fn adhesion forces increased with contact time, emphasizing the important role that time plays in strengthening adhesion.
Force Spectroscopy
Hydrophobic effect
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A novel method is presented for in situ quantification of living cell adhesion forces using a homemade nanorobotic system provided with two independently actuated probes that form a dual-probe nanotweezer capable of pick-and-place manipulation of a single living cell in an aqueous environment. Compared with single-cell force spectroscopy (SCFS) based on traditional atomic force microscopy (AFM), cell immobilization via chemical trapping is unnecessary and the test cell can be efficiently released using the nanotweezer to significantly enhance production of the SCFS. Benefiting from the accurate force sensing capability of AFM, the nanotweezer allows reliable force measurement ranging from picoNewtons to microNewtons and is sufficiently sensitive to characterize short- and long-term adhesion of cell-cell and cell-substrate adhesions. Capabilities of the nanotweezer have been validated through experimental qualification of cell-substrate and cell-cell adhesion events of C2C12 cells (mouse myoblast adherent) with different contact times.
Force Spectroscopy
C2C12
Living cell
Single-Cell Analysis
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Cell to cell interactions can lead to cancer cancerous cells migration and invasion. Atomic force microscopy (AFM) provides a tool that can study the physical properties of nanoscale spatial resolution cells. By attaching individual cells onto the cantilever of AFM, cellular adhesion forces can be quantified, and this technique is called single-cell force spectroscopy (SCFS). In this work, we used AFM-based SCFS to detect adhesion forces between different cells (including cancerous cells and normal cells) and fibronectin, which improves our understanding of cellular adhesion behaviors.
Force Spectroscopy
Cell Mechanics
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In the past 25 years many techniques have been developed to characterize cell adhesion and to quantify adhesion forces. Atomic force microscopy (AFM) has been used to measure forces in the pico-newton range, an experimental technique known as force spectroscopy. We modified such an AFM to measure adhesion forces between live cells or between cells and surfaces. This strategy required functionalizing the surface of the sensors for immobilizing the cell. We used Dictyostelium discoideum cells which respond to starvation by surface expression of the adhesion molecule csA and consequent aggregation to measure the adhesion force of a single csA-csA bond. Relevant experimental parameters include the duration of contact between the interacting surfaces, the force against which this contact is maintained, the number and specificity of interacting adhesion molecules and the constituents of the medium in which the interaction occurs. This technology also permits the measurement of the viscoelastic properties of single cells or cell layers.
Force Spectroscopy
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Objective:The adhesion of cells to cells and cells to stroma play very important role in the cell-cell information exchanges.Methods:The cell adhesion assay based on the 3 H-TdR incorporation assay was introduced. Firstly, coated cells were cultured to confluence in 96 well plate. After incubated with 3 H-TdR for 6h, the isotope labeled cells were added into plate wells and incubated for another 4 h. Then the un-adhered cells were removed by gently washing. The cpm was counted after cell harvest.Results:The cell adhesion assay based on the 3 H-TdR incorporation assay was very sensitive in the detection of cell adhesion. It could reflect the relative of cell to cell adhesion.Conclusion:The cell adhesion assay based on the 3 H-TdR incorporation assay is an useful method in the detection of cell to cell adhesion.
Cell counting
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Abstract A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.
Force Spectroscopy
Characterization
Gelatin
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The use of force spectroscopy to study the adhesion of living fibroblasts to their culture substrate was investigated. Both primary fibroblasts (PEMF) and a continuous cell line (3T3) were studied on quartz surfaces. Using a fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the 3T3 cells from the quartz surfaces. Their adhesion to the quartz surface and the effects of topography on this adhesion could be quantified. Three parameters characteristic of the adhesion were measured: the maximum force of detachment, the work of adhesion, and the distance of detachment. Few PEMF cells were detached under the same experimental conditions. The potential and limitations of this method in measuring cell/surface interactions for adherent cells are discussed.
Force Spectroscopy
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