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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.

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Figure 1: Elastic resonator interference stress microscopy (ERISM).
Figure 2: Validation of ERISM by AFM.
Figure 3: Investigation of podosome protrusions by ERISM.
Figure 4: ERISM for investigation of amoeboid migration in confined space and protein-specific cell–substrate interaction.
Figure 5: Analysis of the horizontal stress applied by adherent 3T3 fibroblasts using ERISM.
Figure 6: ERISM for long-term time-lapse studies of the mechanical activity of cells.

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Acknowledgements

The authors thank K. Venkatesan Iyer and P. A. Reynolds for fruitful discussion, R. Shahapure for TFM reference measurements, A. L. Sobiech for illustrations and the DictyoBase for provision of AX3 strain Dictyostelium discoideum. This project has received funding from the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 640012), the EPSRC DTP (EP/L505079/1), the RS MacDonald Charitable Trust and the MRC (G1100116 and G110312/1).

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Authors

Contributions

N.M.K., P.L. and M.C.G. developed ERISM. N.M.K. fabricated and characterized the micro-cavity substrates and conducted measurements. P.L. developed the data analysis, stress map calculation and graphical data presentation. A.S. contributed to protein coating, staining and cell culture. J.A.K. designed and prepared the primary mouse fibroblast experiment. J.G.B. prepared and assisted in the T cell experiment. G.S. and K.F. performed rheometry and TFM, and contributed to AFM and general discussion. S.J.P. proposed and prepared the macrophage experiment. M.C.G. supervised the project. N.M.K. and M.C.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Malte C. Gather.

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Competing interests

The University of St Andrews has filed a patent application related to the technique described here.

Integrated supplementary information

Supplementary Figure 1 Development and characterization of ERISM micro-cavities and data analysis.

(ad) Oxygen plasma treatment of silicone elastomer surface. (a) Increase in water contact angle for different plasma treatment durations at plasma power of 80–90 W. (b) Reflectance of 15 nm-thick gold layers evaporated on glass (blue) and on untreated elastomer (black). (The oscillation for the black line is due to thin film interference.) (c) Reflectance at 650 nm (left) and apparent Young’s modulus (right, calculated for an indentation force of 2 nN) of surface-oxidized elastomer layers with 15 nm top gold mirror for different plasma durations t and plasma powers P (process pressure, 3 mtorr; gas flow, 20 sccm O2 and 5 sccm Ar). The apparent Young’s modulus was obtained by AFM indentation measurements with a 17 μm-diameter glass sphere. For comparison the reflectance and Young’s modulus, respectively, of a pristine non-oxidized elastomer sample without gold coating are shown at the origins of the tP planes. (d) Plot of apparent Young’s modulus versus reflectance for all samples shown in (c). The region where low reflectance renders ERISM measurements unreliable is marked in red. (e) Wavelength-dependent intensity of the probe light used for the ERISM measurement when incident on the bottom glass side of the micro-cavity through a 4×, 10×, 20× and 40× objective, respectively. (f) Measurement of the Young’s modulus of the elastomer within the micro-cavity obtained by comparing AFM indentation forces to FEM predictions obtained from ERISM deformation profiles. See Methods for details.

Supplementary Figure 2 Flow cytometry of isolated human macrophages.

Adherent cells from isolated peripheral blood mononuclear cells were recovered and stained with anti-CD14-PE, shown shaded, compared to auto-fluorescence from cells without antibody staining (unshaded).

Supplementary Figure 3 Demonstration of the sensitivity of ERISM to horizontally applied forces.

(a) The needle of an AFM cantilever is slightly indented into the ERISM chip to make firm contact and the cantilever is then pulled to the side to apply a defined lateral force. This gives rise to an asymmetric ERISM signal that grows with increasing horizontal force. (b) Peak ERISM signal detected versus horizontal force applied by AFM. Red line, linear fit (R2 > 0.99). Dashed lines, displacement resolution of ERISM in the present configuration and resulting minimal horizontal force that can be detected.

Supplementary Figure 4 High frame rate ERISM (ab), ERISM for long-term time-lapse studies of the mechanical activity of cells during mesenchymal migration (ce) and differentiation of fibroblasts into myofibroblasts in situ on the ERISM micro-cavity (fg).

(a) Phase contrast image and ERISM map of 3T3 fibroblast cells. Images from an 11.5 min-ERISM time-lapse measurement with a frame acquired every 2.3 s. (b) Evolution of mean displacement under the cell nucleus and the lamellum, respectively (areas circled in blue and red in (a)). The black trace shows the evolution for an area where no cell is present (black circle in (a)). Also see Supplementary Video 5. (c) Phase contrast image and ERISM map for primary mouse fibroblasts after cells migrated out of a tissue explant directly placed on the micro-cavity substrate. (d) ERISM time-lapse series of fibroblast migration for the cell highlighted in (c). The cell boundary is indicated by a black line. (e) Temporal evolution of (left) the force applied in each direction with respect to the cell centre as defined in the first panel of (d) and (right) the total force and speed of cell migration. Position and size of white circles indicate direction and speed of migration. [A similar analysis of the moment of the force orientation forces has been performed for Dictyostelium discoideum using TFM, see H. Tanimoto and M. Sano, Biophys. J. 106, 16–25 (2014).] (f) Representative phase contrast image (left) and ERISM stress map (right) of a cell after 66 h of TGF-β treatment. Cell outline is shown in black. (g) Fluorescence images of immune-labelled α-SMA (green) after fixation and permeabilisation of cells on the micro-cavity. Left: Cells stimulated with TGF-β for 66 h. Right: Non-stimulated control cells kept on the micro-cavity for the same time. While cells in the control group show weak and unspecific background fluorescence, parallel arranged α-SMA fibres arching through the cell bodies are clearly visible in differentiated cells. Scale bars, (a) 50 μm, (c) 100 μm, (d,f) 50 μm and (g) 200 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 753 kb)

Illustration of mechanical stability of the micro-cavities used for ERISM.

Real time video of monochromatic reflectance of a micro-cavity, into which a 17 μm dimeter glass bead (glued to the tip of an AFM probe) was indented by 900 nm. While keeping the indentation constant, the bead is moved horizontally across the micro-cavity. (AVI 5296 kb)

Fourier-filtered ERISM time-lapse investigation of podosome protrusions of a human macrophage.

Phase contrast microscopy (left) and spatial Fourier-filtered stress map (right). (AVI 7940 kb)

Fourier-filtered ERISM time-lapse investigation of podosome protrusion of a human macrophage during cell migration.

Phase contrast microscopy (top) and spatial Fourier filtered substrate displacement (bottom). (AVI 4644 kb)

ERISM time-lapse investigation of amoeboid migration in spatial confinement.

Video shows the substrate displacement caused by a Dictyostelium discoideum amoeba migrating through a 5 μm-thick void formed above the micro-cavity substrate. The outline of the amoeba is shown in black. (AVI 1899 kb)

High-speed ERISM time-lapse investigation of 3T3 fibroblasts.

Phase contrast microscopy (left) and substrate displacement in nm (right). The inset shows the displacement for the region highlighted by a white box in the bright-field and displacement images with increased contrast to show displacements due to expansion of the cell edge in more detail. A frame was taken every 2.3 s. (AVI 2699 kb)

Long-term ERISM time-lapse investigation of 3T3 fibroblasts.

Phase-contrast microscopy (left) and substrate displacement in nm (right). The video was recorded over 5.5 days and contains more than 1,600 individual ERISM maps. (AVI 15341 kb)

ERISM time-lapse investigation of mesenchymal migration in a culture of primary mouse fibroblasts.

Video shows an overlay of substrate displacement with corresponding phase contrast microscopy image. Pulling is indicated in red, pushing is indicated in blue. (AVI 7125 kb)

Close-up ERISM time-lapse of mesenchymal migration of individual primary mouse fibroblast.

Phase contrast microscopy (left) and substrate displacement (right). (AVI 642 kb)

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Kronenberg, N., Liehm, P., Steude, A. et al. Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy. Nat Cell Biol 19, 864–872 (2017). https://doi.org/10.1038/ncb3561

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