Neuron biomechanics probed by atomic force microscopy

Abstract

Mechanical interactions play a key role in many processes associated with neuronal growth and development. Over the last few years there has been significant progress in our understanding of the role played by the substrate stiffness in neuronal growth, of the cell-substrate adhesion forces, of the generation of traction forces during axonal elongation, and of the relationships between the neuron soma elastic properties and its health. The particular capabilities of the Atomic Force Microscope (AFM), such as high spatial resolution, high degree of control over the magnitude and orientation of the applied forces, minimal sample damage, and the ability to image and interact with cells in physiologically relevant conditions make this technique particularly suitable for measuring mechanical properties of living neuronal cells. This article reviews recent advances on using the AFM for studying neuronal biomechanics, provides an overview about the state-of-the-art measurements, and suggests directions for future applications.

Figure 1

(a) Schematic of Atomic Force Microscope (AFM) cantilever holder and laser detector setup. Before calibration, tip deflection is measured as a voltage value generated by a difference in light falling on the upper vs. lower quadrants of the photo sensor. In order to measure cantilever deflection as a distance, the cantilever must be calibrated. This is typically done by performing a force curve on an “infinitely” hard surface (zero indentation), and equating the decrease in cantilever z height after contact with the increase in tip deflection [5]; (b) AFM topography image of ridges on a micropatterned silicon substrate shown as an example of a representative AFM image. Image taken by Elise Spedden in Staii lab at Tufts University.

Figure 2

(a) Typical AFM force vs. indentation curve. The contact point is defined as the point at which the force vs. indentation slope begins to increase above noise level. The flat line indicates the approach portion, where the cantilever is moving down but it is not yet in contact with the surface. The curved portion after the contact point indicates how the total force of the tip on the sample changes as the cantilever indents the surface; (b) Optical image of living cortical neuron; (c) Neuron shown in (a) with superimposed 1 × 1 micron grid; (d) Elastic modulus map of neuron from (c), where force vs. indentation curves are taken at each point on the grid and elastic modulus values are calculated from the indentation curves (see text). These images illustrate the elasticity mapping procedure where optical and AFM measurements are taken simultaneously. Images taken by Elise Spedden, in Staii lab at Tufts University.

Figure 3

(a) Example of a 3D AFM topography image of a live cortical neuron soma; (b) 3D AFM topography image of a live neuron growth cone. Both images were taken in force-volume mode, thus rendering lower spatial resolution than high-resolution AFM topography in tapping mode. However, force-volume mode images contain both topographical and elasticity information as explained in the main text. Images taken by Elise Spedden in Staii lab at Tufts University.

Figure 4

(a) Topography of live neuron soma obtained during elastic modulus mapping; (b) High resolution elastic modulus map of cell shown in (a). The cell displays regions of high elastic modulus localized at the lower end (bright areas in the figure). Images taken by Elise Spedden in Staii lab at Tufts University.

Figure 5

(a) Fluorescent image of a living neuron soma stained for tubulin density; (b) Elastic modulus map of cell shown in (a). The images have the same scale bar shown in (b). There is a significant overlap between areas of high actin concentration (high fluorescence intensity) and the regions of high stiffness. Images taken by Elise Spedden in Staii lab at Tufts University.