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Description
The atomic force microscope (AFM) is capable of directly probing the mechanics of samples with length scales from single molecules to tissues and force scales from pico to micronewtons. In particular, AFM is widely used as a tool to measure the elastic modulus of soft biological samples by collecting force-indentation relationships and fitting these to classic elastic contact models. However, the analysis of raw force-indentation data may be complicated by mechanical heterogeneity present in biological systems. An analytical model of an elastic indentation on a bonded two-layer sample was solved. This may be used to account for substrate effects and more generally address experimental design for samples with varying elasticity. This model was applied to two mechanobiology systems of interest. First, AFM was combined with confocal laser scanning fluorescence microscopy and finite element analysis to examine stiffness changes during the initial stages of invasion of MDA-MB-231 metastatic breast cells into bovine collagen I matrices. It was determined that the cells stiffen significantly as they invade, the amount of stiffening is correlated with the elastic modulus of the collagen gel, and inhibition of Rho-associated protein kinase reduces the elastic modulus of the invading cells. Second, the elastic modulus of cancer cell nuclei was investigated ex situ and in situ. It was observed that inhibition of histone deacetylation to facilitate chromatin decondenstation result in significantly more morphological and stiffness changes in cancerous cells compared to normal cells. The methods and results presented here offer novel strategies for approaching biological systems with AFM and demonstrate its applicability and necessity in studying cellular function in physiologically relevant environments.
ContributorsDoss, Bryant Lee (Author) / Ros, Robert (Thesis advisor) / Lindsay, Stuart (Committee member) / Nikkhah, Mehdi (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2015
Description
Mechanical properties, in particular elasticity, of cancer cells and their microenvironment are important in governing cancer cell fate, for example function, mobility, adhesion, and invasion. Among all tools to measure the mechanical properties, the precision and ease of atomic force microscopy (AFM) to directly apply force—in the range of Pico to micronewtons—onto samples—with length scales from nanometers to tens of micrometers—has made it a powerful tool to investigate the mechanics of materials. AFM is widely used to measure deformability and stiffness of soft biological samples. Principally, these samples are indented by the AFM probe and the forces and indentation depths are recorded. The generated force-indentation curves are fitted with an elastic contact model to quantify the elasticity (e.g. stiffness). AFM is a precise tool; however, the results are as accurate as the contact model used to analyze them. A new contact model was introduced to analyze force-indentation curves generated by spherical AFM probes for deep indentations. The experimental and finite element analysis results demonstrated that the new contact model provides more accurate mechanical properties throughout the indentation depth up to radius of the indenter, while the Hertz model underestimates the mechanical properties. In the classical contact models, it is assumed that the sample is vertically homogenous; however, many biological samples—for example cells—are heterogeneous. A novel two-layer model was utilized to probe Polydimethylsiloxane hydrogel (PDMS) layers on PDMS substrates with stiffness mismatch. In this experiment the stiffness of the substrate was deconvoluted from the AFM measurements to obtain the stiffness of the layer. AFM and confocal reflectance microscopy were utilized along with a novel 3D microengineered breast cancer tumor model to study the crosstalk between cancer tumor and the stromal cells (CAFs) and the ECM remodeling caused by their interplay. The results showed that as the cancer cells invade into the extracellular matrix (ECM), they release PDGF ligands which enable Cafes to remodel the ECM and this remodeling increased the invasion rate of the cancer cells. Next, the effect of the ECM remodeling on anti-cancer drug resistant was investigated within the 3D microengineered cancer model. It was demonstrated that the combinatory treatment by anti-cancer and-anti-fibrotic drugs enhance the efficiency of the cancer treatment. A novel DNA-based 3D hydrogel model with tunable stiffness was investigated by AFM. The results showed the hydrogel stiffness can be enhanced by adding DNA crosslinkers. In addition, the stiffness was reduced to the control sample level by introducing the displacement DNA. Biophysical quantifications along with the in vitro microengineered tumor models provide a unique frame work to study cancer in more detail.
ContributorsRahmani Eliato, Kiarash (Author) / Ros, Robert (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Ozkan, Sefika (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2019