Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy

Abstract

Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research.

Keywords: (100.6950) Tomographic image processing; (170.1530) Cell analysis; (170.4500) Optical coherence tomography; (170.6900) Three-dimensional microscopy.

Fig. 1

Measurement noise floor of the DIC-based ECM displacement tracking algorithm (described in Section 2.6) in the (a) xy-plane and (b) zx-plane, respectively. The variable ‘correlation window side length’ refers to the side length of the windowed deformed-state image. The different curves represent the use of median filters of different sizes following the cross-correlation operation. Displacement noise floors corresponding to the final parameters used in the tracking of cell-induced displacements are denoted by black arrows.

Fig. 2

Automated tracking of 3D deformations induced by NIH-3T3 fibroblasts cultured in a Matrigel-derived ECM. These images represent the deformations accumulated over a 90 minute imaging time, with reagents introduced to the sample after the first 30 minutes of imaging. Cells were exposed to pure DMSO (a-c), or cytochalasin D solution (d-f). Each subfigure depicts displacements in the en face (upper panels) and vertical (lower panels) orientations. (a,d) Superposition of the initial (t = 0 minutes, red channel) and final (t = 90 minutes, green channel) states of the sample, obtained from the initial and final registered maximum intensity projection images described in Section 2.4. (b,e) Cumulative displacement magnitude of the extracellular matrix (in µm) from a given initial location. (c,f) Cumulative displacement field depicting the direction and relative magnitude of ECM displacement (with arrow lengths exaggerated for visibility), superimposed on the initial maximum intensity projection images. Scale bars = 50 µm. Refer to the text for a discussion of the arrows in (a), (d), and (e).

Fig. 3

Automated and manual tracking of embedded polystyrene bead cumulative displacement magnitudes in time (top) at varying locations around the cells (bottom) exposed to (a) pure DMSO, or (b) cytochalasin D dissolved in DMSO. All displacement magnitudes are defined with respect to the initial location of a given bead. Solid curves depict results of manual single particle tracking; dashed curves depict results of automated DIC-based displacement tracking. The vertical dashed lines in the displacement plots mark the time at which the DMSO or cytochalasin D was added to the samples. Scale bars = 50 µm.

Fig. 4

Automated and manual tracking of cumulative displacement magnitudes undergone by embedded polystyrene beads at various selected locations around fibroblasts exposed to the control conditions (DMSO). Each subplot (a-h) depicts the results obtained from independent trials of the experimental protocol. The first subplot (a) depicts the same data discussed in Fig. 3(a). All displacement magnitudes are defined with respect to the initial location of a given bead. Solid curves depict results of manual single particle tracking; dashed curves depict results of automated DIC-based displacement tracking. The vertical dotted lines mark the time at which the samples were exposed to DMSO.

Fig. 5

Automated and manual tracking of cumulative displacement magnitudes undergone by embedded polystyrene beads at various selected locations around fibroblasts exposed to the contractility inhibiting conditions (cytochalasin D + DMSO). Each subplot (a-h) depicts the results obtained from independent trials of the experimental protocol. The first subplot (a) depicts the same data discussed in Fig. 3(b). All displacement magnitudes are defined with respect to the initial location of a given bead. Solid curves depict results of manual single particle tracking; dashed curves depict results of automated DIC-based displacement tracking. The vertical dotted lines mark the time at which the samples were exposed to cytochalasin D solution.