Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy

Abstract

Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to data analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell-substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic resonator interference stress microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 μm range, translating into 1 pN force sensitivity). This enables investigation of minute vertical stresses (<1 Pa) involved in podosome protrusion, protein-specific cell-substrate interaction and amoeboid migration through spatial confinement in real time. ERISM requires no zero-force reference and avoids phototoxic effects, which facilitates force monitoring over multiple days and at high frame rates and eliminates the need to detach cells after measurements. This allows observation of slow processes such as differentiation and further investigation of cells, for example, by immunostaining.

Cortical cell stiffness is independent of substrate mechanics

“…First, maps of the vertical substrate deformation caused by the contractility of the cell were recorded by imaging the reflectance of the ERISM substrate at 201 different wavelengths between 550 and 750 nm as described previously. 18 Next, the AFM cantilever was lowered onto the center of the cell until a compression force of 0.5 nN was reached. The force was kept constant via a feedback loop while repeating the ERISM readout using a reduced wavelength range of 51 nm to accelerate the measurement (<5 s) 19 .…”

“…The force applied by the cantilever onto the cell is balanced by the elastic deformation of the substrate underneath the cell (i.e., the force experienced by the substrate is the same as that exerted onto the cell). To investigate this substrate deformation in more detail, we combined AFM with Elastic Resonator Interference Stress Microscopy (ERISM) 17,18 , which quantifies the vertical deformation of deformable substrates with high spatial resolution ( Fig. 2).…”

“…1 for details Elastic resonator interference stress microscopy. ERISM substrates with an apparent stiffness of 3 kPa were fabricated as described previously 17 . A silicon chamber (surface area:…”

“…Combined ERISM-AFM measurements were carried out in cell medium at 37 °C. First, maps of the vertical substrate deformation caused by the contractility of the cell were recorded by imaging the reflectance of the ERISM substrate at 201 different wavelengths between 550 and 750 nm as described previously 17 . Next, the AFM cantilever was lowered onto the center of the cell until a compression force of 0.5 nN was reached.…”

“…To investigate the vertical displacement field of the substrate under cells caused by AFM indentation measurements in more detail, we combined AFM with Elastic Resonator Interference Stress Microscopy (ERISM) 18,19 , which quantifies the vertical deformation of deformable substrates with high spatial resolution ( Figure 2). Both the substrate deformation and the stress were maximum under the cell center, where the cantilever was located, and increased linearly with the applied force (Figure 2a).…”