High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy

Abstract

A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.

Fig. 1. Layout of refractive-index traction force microscopy (RI-TFM).

a Schematic. We implemented RI tomography using an off-axis holographic microscope to image a live cell on an elastic polyacrylamide hydrogel embedding 200-nm-diameter polystyrene microspheres. The schematic diagram of the optical setup is illustrated in Supplementary Fig. 1. b XZ- and XY-sliced FL and RI images of a fibroblast cell expressing paxillin-EGFP, which was mounted on an 11-kPa hydrogel. c Postprocessing. The hydrogel was imaged twice: before (magenta) and after (cyan), the cell was chemically detached using an EDTA solution. The acquired tomograms were high-pass filtered, masked, and registered. Inset images show magnified regions where 3D displacements of the embedded beads were significant. d 3D displacement vectors were computed using a correlation method based on the Fourier transform and drawn in the maximum RI projections of the cell. RMSE root-mean-squared error. e Estimated multidimensional traction of the cell using RI-TFM was compared with that of FL-TFM.

Fig. 2. Accuracy and precision of RI-TFM.

a Representative fluorescence and RI tomograms. 3D displacement vectors were estimated when the hydrogel was laterally displaced by 3 μm. Arrows denote the estimated displacement vectors at each spacing grid. b Statistics of measured displacement vectors. Points and horizontal lines indicate the mean and mean ± standard deviation, respectively. The number of data points per statistic is >2700. The dashed line represents the expected linear plot. c Measurement sensitivity of multidimensional traction stress per Young’s modulus was defined as its standard deviation. σ standard deviation, RMSE root mean squared error.

Fig. 3. 3D RI and traction of NIH-3T3 fibroblasts expressing paxillin-EGFP before and after treatment of cytochalasin D.

a Representative images of overlaid 3D-rendered tomograms of RI (grey) and FL (paxillin-EGFP; green) and traction stress for NIH3T3 cell 1 (top) and 2 (below) before and 1 h after treatment with cytochalasin D (5 μM). For clearer visualization, we down-sampled the traction vectors and drew significant traction vectors above threshold values in the XY images. b Histograms of pressure by weight, shear traction, and normal traction before (magenta) and after (cyan) cytochalasin D treatment. The mean ± standard deviation for each histogram is presented.

Fig. 4. Sequential images of the MDCK cell detached from an 11-kPa gel substrate.

a 3D-rendered time sequences. Vectors indicate the differential traction stress, ΔT_j = T_j – T_j-1, where j is the frame index. Here, we defined T_{0 s} = 0. b XZ maximum intensity projections, XY surface dry-mass distributions, and multidimensional traction stress maps of selected sequences in (a) (red). White arrows indicate regions related to the detached focal adhesion. For clearer visualization, significant traction vectors above threshold values were drawn in the XY images. c, d Kymographs along the x direction for (c), 3D morphological parameters (maximum cell thickness and average surface dry-mass density), d maximal value for the differential shear and normal traction. See Supplementary Videos 2 and 3 for more details.

Fig. 5. Quantification of the multidimensional traction forces generated by human CD8+ cytotoxic T lymphocytes (CTLs).

a, b, Representative sequential snapshots of (a) resting and (b) activated CTL on a 1.2-kPa gel coated with anti-CD3/CD28 antibodies. For clearer visualization, significant traction vectors above threshold values were drawn in the XY images. c Temporal changes in normal traction, shear traction (second row), and surface dry-mass densities for resting and activated CTLs in (a, b). The central points and shades indicate the mean and standard deviation (number of points > 50), respectively. d, Representative sequential snapshots of activated CTLs acquired at an interval of 2 s. See Supplementary Videos 4 and 5 for more details.