Force Sensing on Cells and Tissues by Atomic Force Microscopy

Abstract

Biosensors are aimed at detecting tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM) and the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, cells themselves also use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influence cell fate, regulate tissue development, and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissecting and understanding complex biological processes, such as mechanotransduction. Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanonewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology. In this work, we report on the application of AFM to the study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, such as the nucleus, lamellipodia and the glycocalyx. We show that physical observables such as the (spatially resolved) Young's Modulus (YM) of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterized by AFM. Their modification can be correlated to changes in the microenvironment, physio-pathological conditions, or gene regulation.

Keywords: Atomic Force Microscopy; biosensors; colloidal probe; extracellular matrix; glycocalyx; mechanobiology.

Figure 1

(A) Scheme of the optical beam deflection (OBD) system. The vertical displacement of the cantilever induced by the sensing of a force F perpendicular to the sample surface is detected on a segmented photodiode as a raw voltage signal $\Delta \mathrm{V}$. The cantilever is typically mounted at an angle $\mathsf{\theta }$ with respect to the sample surface. (B) A raw force curve, representing the photodiode output $\Delta \mathrm{V}$ as a function of the z-piezo displacement ${\mathrm{d}}_{\mathrm{p}}$. Both the approaching and retracting branches of the curve are shown.

Figure 2

Typical approaching force curve on top of a cell (top) and ECM (bottom). The indentation range for model fitting is highlighted; on cells, we used typically [0–10%] for the glycocalyx and [10–80%] for the YM, while on ECMs we used [20–80%] for the Young’s Modulus.

Figure 3

(A) Schematics of the distances used to determine the tip-cell membrane distance H (Equation (8)). (B) A typical force curve showing the force exerted by the glycocalyx as a function of the tip-cell membrane distance (the red continuous curve is the fit by Equation (9)).

Figure 4

The stiffening of ECM in CRCPM samples. (A) Logarithmic values of the YM and their distribution in normal and neoplastic ECM samples obtained from one patient. Violin plots were plotted collecting YM values from all single FCs. Violin plots suggest that the distribution of local YM values is approximately lognormal. The circle and the black bars represent the median and the interval between 25th and 75th percentiles. (B) Comparison of median YM values E_med from each force volume in linear scale for normal and neoplastic samples from one patient. The red line represents the median value, the box encloses the interval between 25th and 75th percentiles of the sample. Whiskers go from the upper and lower limits of the interquartile range to the furthest observations, within 1.5× the interquartile range; data points beyond this limit are considered outliers. (C) Comparison of the mean median YM values for the two conditions tested. In (A, C), * means p < 0.05.

Figure 5

Collagen of ECM of healthy (A) and tumoral region (B) stained with van Gieson trichrome.

Figure 6

Representative images for the combined topographical and mechanical analysis of cell clusters. Optical image of a cell cluster from the RT112 cell line (A); topographic map (B), and Young’s modulus map (C) in logarithmic scale, of the same cluster shown in (A).

Figure 7

Histograms of the YM from the perinuclear and peripheral region of an RT112 cell (orange), and from the nuclear region (red).

Figure 8

The Young’s modulus (whole cell) measured by AFM for bladder cancer cells RT4, RT112, and T24, with increasing grades of invasiveness (left to right). * Means p < 0.05.

Figure 9

Distribution of the lengths of the glycocalyx brush (extracted according to Equation (9) from single FCs) for the three cell lines RT4, RT112, and T24. Vertical dotted lines are a guide for the eye in the tentative identification of the main modes of the distributions.