Holographic Traction Force Microscopy

Abstract

Traction Force Microscopy (TFM) computes the forces exerted at the surface of an elastic material by measuring induced deformations in volume. It is used to determine the pattern of the adhesion forces exerted by cells or by cellular assemblies grown onto a soft deformable substrate. Typically, colloidal particles are dispersed in the substrate and their displacement is monitored by fluorescent microscopy. As with any other fluorescent techniques, the accuracy in measuring a particule’s position is ultimately limited by the number of evaluated fluorescent photons. Here, we present a TFM technique based on the detection of probe particle displacements by holographic tracking microscopy. We show that nanometer scale resolutions of the particle displacements can be obtained and determine the maximum volume fraction of markers in the substrate. We demonstrate the feasibility of the technique experimentally and measure the three-dimensional force fields exerted by colorectal cancer cells cultivated onto a polyacrylamide gel substrate.

Where No Hand Has Gone Before: Probing Mechanobiology at the Cellular Level

“…A state-of-the-art TFM technique that offers higher force resolution than conventional TFM is holographic TFM . Holographic TFM, which was recently developed by Makarchuk and colleagues, uses sparse nonfluorescent particles instead of the conventional high density of fiducial markers.…”

“…Confocal microscopy can be used to track the position of the fiducial markers in 3D during conventional TFM, − as well as Quantum Dot TFM, to resolve 3D deformation fields from which the shear and normal forces can be reconstructed. Some of the techniques presented here, such as holographic TFM, can also resolve out-of-plane forces without the need for confocal microscopy …”

Epifluorescence-based three-dimensional traction force microscopy

“…Makarchuk et al . used holographic traction force microscopy to localize and track fiducial particles embedded within the substrate from phase contrast images 38 . In a similar technique, Hall et al .…”

Physicochemical Tools for Visualizing and Quantifying Cell-Generated Forces

“…Another recent approach to high-resolution TFM is the use of holographic tracking microscopy to measure the displacements of fiducial markers. 40 Known as holographic TFM, the technique can achieve nanometer-scale resolutions of marker displacements through the use of nonfluorescent particles at low spatial density (Figure 4, panel c). The technique relies on analyzing the diffraction patterns created by an LED light source shone on micron-sized polystyrene particles.…”

“…Moreover, by taking a stack of the sectioned images in the z dimension, the technique can be used to quantify normal forces generated by the cell and, thus, reconstruct 3D stress fields at both high spatial and temporal resolution. Another recent approach to high-resolution TFM is the use of holographic tracking microscopy to measure the displacements of fiducial markers . Known as holographic TFM, the technique can achieve nanometer-scale resolutions of marker displacements through the use of nonfluorescent particles at low spatial density (Figure , panel c).…”