High Resolution Mapping of Cytoskeletal Dynamics in Neurons via Combined Atomic Force Microscopy and Fluorescence Micros
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High Resolution Mapping of Cytoskeletal Dynamics in Neurons via Combined Atomic Force Microscopy and Fluorescence Microscopy Elise Spedden1, David L. Kaplan2, 3, Cristian Staii1 1 Department of Physics and Astronomy, Tufts University, 4 Colby St, Medford Ma 02155 2 Department of Biomedical Engineering, Tufts University, 4 Colby St, Medford Ma 02155 3 Department of Chemical Engineering, Tufts University, 4 Colby St, Medford Ma 02155 ABSTRACT Living neuronal cells present active mechanical structures which evolve with cellular growth and changes in the cell microenvironment. Detailed knowledge of various mechanical parameters such as cell stiffness or adhesion forces and traction stresses generated during axonal extension is essential for understanding the mechanisms that control neuronal growth, development and repair. Here we present a combined Atomic Force Microscopy (AFM)/Fluorescence Microscopy approach for obtaining systematic, high-resolution elasticity and fluorescent maps for live neuronal cells. This approach allows us to simultaneously image and apply controllable forces to neurons, and also to monitor the real time dynamics of the cell cytoskeleton. We measure how the stiffness of neurons changes both during axonal growth and upon chemical modification of the cell, and identify the cytoskeletal components most responsible for the changes in cellular elasticity. This is accomplished by identifying cellular components with unique elastic signatures, and tracking those components over time within healthy cells or within cells treated to disrupt selective components. INTRODUCTION The Hertz model as applied to AFM measurements has been used to measure elasticity of living and fixed cells for over 15 years1. Over time the methods have evolved allowing more detailed and accurate information to be extracted2-4. The field of elasticity measurements has been growing, but many aspects of understanding still remain largely unexplored. Studies in AFM elasticity of living cells have pointed to a link between soma elasticity values and cell health 5. Neuronal cells in particular have shown a link between the elasticity of the soma and the stiffness of the growth substrate 6, 7. The physical properties of the cell microenvironment are important for growth and directionality 8, 9. Understanding the mechanical properties of cells, and neurons in particular, helps with understanding how these cells might respond to mechanical stimuli or navigate over stiffer or weaker environments. Different types of neurons exhibit distinct elastic properties6, 7, They also show preferential growth over substrates of well-defined stiffness and produce different traction forces during growth 6, 7. The level of internal stiffness is likely to correlate with the cell’s ability to generate traction forces, an important factor for axonal growth and navigation. The ability to measure cell elasticity also allows the measurement and tracking of elastically identifiable components and affords the opportunity to monitor changes to these components ov
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