Elasticity Maps of Living Neurons Measured by Combined Fluorescence and Atomic Force Microscopy

Abstract

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain

“…(2) The majority (92-97%) of cells are in the neural lineage ( Table 1). A previous report has indicated that the stabilization of microtubules contributes to the increase in the stiffness of a cortical neuron (Spedden et al, 2012), raising the possibility that the stiffness of neural cells is determined by the status of the microtubule cytoskeleton. This notion is supported by the increasing amount of tubulin β III (Tuj1) in the soma during neural maturation (Fig.…”

“…The brain is one of the softest tissues in the human body (Moore et al, 2010;Spedden et al, 2012). Its stiffness in the postnatal stage has been examined previously in the rodent cerebral cortex (Elkin et al, 2010), the hippocampus (Elkin et al, 2007) and the cerebellum (Christ et al, 2010), although it has never been measured at developing stages (Franze, 2013).…”

Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth

“…On the other hand, viability assay and fluorescent image analysis (Figure 7) identified that the neuron adhesion was significantly higher The modulus of elasticity for the neurons is been reported to be between 3 to 6 KPa 45,46 . The modulus of our membrane tested using Nano indentation was found to be about 150 to 350 MPa.…”

Neuronal growth as diffusion in an effective potential

“…These studies show that physical stimuli (gradients of various molecular species, stiffness of the growth substrate, traction forces generated during axonal extension etc.) play a key role in the wiring of the nervous system [3][4][5][6][7][8][9]. However, our current understanding of neuronal growth is mostly qualitative, the vast complexity of the parameter space still prohibiting fully quantitative predictions of outcomes from given initial conditions such as: geometry of the neuronal circuit, type of biochemical cues on the growth substrate, topography or mechanical properties of the substrate.…”

“…After plating, cells were allowed to incubate at 37°C for 8, 15, 19, 26, 33, or 46 hours. Samples are then removed from the incubator, loaded into the BioHeater™ Closed Fluid Cell, and imaged under bright-field using the optical stage of the MFP-3D-BIO Atomic Force Microscope (AFM, Asylum Research) [9,19] (Fig. 1).…”