Combined Traction Force–Atomic Force Microscopy Measurements of Neuronal Cells

Abstract

In the course of the development of the nervous system, neuronal cells extend (grow) axons, which navigate over distances of the order of many cell diameters to reach target dendrites from other neurons and establish neuronal circuits. Some of the central challenges in biophysics today are to develop a quantitative model of axonal growth, which includes the interactions between the neurons and their growth environment, and to describe the complex architecture of neuronal networks in terms of a small number of physical variables. To address these challenges, researchers need new experimental techniques for measuring biomechanical interactions with very high force and spatiotemporal resolutions. Here we report a unique experimental approach that integrates three different high-resolution techniques on the same platform—traction force microscopy (TFM), atomic force microscopy (AFM) and fluorescence microscopy (FM)—to measure biomechanical properties of cortical neurons. To our knowledge, this is the first literature report of combined TFM/AFM/FM measurements performed for any type of cell. Using this combination of powerful experimental techniques, we perform high-resolution measurements of the elastic modulus for cortical neurons and relate these values with traction forces exerted by the cells on the growth substrate (poly acrylamide hydrogels, or PAA, coated with poly D-lysine). We obtain values for the traction stresses exerted by the cortical neurons in the range 30–70 Pa, and traction forces in the range 5–11 nN. Our results demonstrate that neuronal cells stiffen when axons exert forces on the PAA substrate, and that neuronal growth is governed by a contact guidance mechanism, in which axons are guided by external mechanical cues. This work provides new insights for bioengineering novel biomimetic platforms that closely model neuronal growth in vivo, and it has significant impact for creating neuroprosthetic interfaces and devices for neuronal growth and regeneration.

Atomic force microscopy in disease‐related studies: Exploring tissue and cell mechanics

“…Additionally, the amalgamation with deformability cytometry enhances the accuracy of high‐throughput quantification of cellular mechanics (Lei et al, 2021). Furthermore, when combined with traction force microscopy (TFM), it elucidates that neuronal cell axons exert traction forces on the substrate while cell stiffness increases (Kumarasinghe et al, 2022). The coalescence of both methods elucidates the transition of astrocytes and glioblastomas from a non‐migratory (solid‐like) to a migratory (liquid‐like) state through quantifying cell–cell adhesion and tension (Hohmann et al, 2023).…”

Impact of Silk-Ionomer Encapsulation on Immune Cell Mechanical Properties and Viability

“…We used NanoWorld Pyrex-Nitride triangular (PNP-TR) AFM probes (Asylum Research, Santa Barbara, California) possessing a force constant of 0.08 N/m. The value of the elastic modulus at each point on the sample was determined by fitting the force vs indentation curves. , …”

“…Quantitative measurements of material stiffness can be obtained by fitting the force–indentation curve to the corresponding mechanical model. This technique has been widely used to characterize the mechanical properties of living cells and tissues. − Using the AFM nanoindentation technique, we investigated the variation of stiffness of cells after the encapsulation process with different numbers of bilayers. Furthermore, we compared our experimental data for stiffness measured with the AFM with the predictions of a mathematical model based on the Bec/Tonck framework .…”

Combining atomic force microscopy with complementary techniques for multidimensional single‐cell analysis

“…The experimental results directly reveal the correlation between cell viscoelasticity and cell traction force: stiffer cells have a lower fluidity and a larger prestress than softer cells. In 2022, Kumarasinghe et al 192 . combined AFM with TFM to investigate the multidimensional mechanics of neurons.…”

“…The experimental results directly reveal the correlation between cell viscoelasticity and cell traction force: stiffer cells have a lower fluidity and a larger prestress than softer cells. In 2022, Kumarasinghe et al 192 combined AFM with TFM to investigate the multidimensional mechanics of neurons. High-resolution measurements of the Young's modulus of cortical neurons were performed by AFM, which were correlated with traction forces exerted by the cells measured by TFM, showing that neuronal cells stiffen when axons exert forces on the PAA substrate.…”