Epifluorescence-based three-dimensional traction force microscopy

Abstract

We introduce a novel method to compute three-dimensional (3D) displacements and both in-plane and out-of-plane tractions on nominally planar transparent materials using standard epifluorescence microscopy. Despite the importance of out-of-plane components to fully understanding cell behavior, epifluorescence images are generally not used for 3D traction force microscopy (TFM) experiments due to limitations in spatial resolution and measuring out-of-plane motion. To extend an epifluorescence-based technique to 3D, we employ a topology-based single particle tracking algorithm to reconstruct high spatial-frequency 3D motion fields from densely seeded single-particle layer images. Using an open-source finite element (FE) based solver, we then compute the 3D full-field stress and strain and surface traction fields. We demonstrate this technique by measuring tractions generated by both single human neutrophils and multicellular monolayers of Madin–Darby canine kidney cells, highlighting its acuity in reconstructing both individual and collective cellular tractions. In summary, this represents a new, easily accessible method for calculating fully three-dimensional displacement and 3D surface tractions at high spatial frequency from epifluorescence images. We released and support the complete technique as a free and open-source code package.

Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix

“…Traction forces generated by cells lead to matrix deformation, enabling force transmission to distant cells. In a 3D scenario, traction forces generated by a single cell or spheroid can be well determined by traction force microscopy, which measures the displacement of microbeads evenly distributed in the matrix (36,37). We observed that collagen fibers were aligned between two SK-MEL-5 cells.…”

Light-sheet scattering microscopy to visualize long-term interactions between cells and extracellular matrix

“…Traction forces generated by cells lead to matrix deformation, enabling force transmission to distant cells. In a 3D scenario, traction forces generated by a single cell or spheroid can be well determined by traction force microscopy, which measures the displacement of microbeads evenly distributed in the matrix [28,29]. We observed that collagen fibers were aligned between two SK-MEL-5 cells.…”

“…Traction forces generated by cells lead to matrix deformation, enabling force transmission to distant cells. In a 3D scenario, traction forces generated by a single cell or spheroid can be well determined by traction force microscopy, which measures the displacement of microbeads evenly distributed in the matrix [21,22]. For cell-cell distant mechanical communication, where two distantly located cells simultaneously apply forces on matrix mesh network, the displacement of the beads per se might not be suitable to reflect the forces applied as in the case for tug-of-war.…”

SerialTrack: ScalE and Rotation Invariant Augmented Lagrangian Particle Tracking

“…This is in contrast to digital image/volume correlation (DIC/DVC) and particle image velocimetry (PIV), which are subset-correlation-based techniques (see a summary of various full-field tracking methods in Table 2). The increased specificity of the tracked particles can be beneficial to applications such as quantitative biological motion tracking [4,5,6,7,8,9] and fluid mechanics for flow measurements [10,2,11,12]. However, it remains a challenge to uniquely and robustly match particles throughout an image sequence.…”