Multiple‐Particle Tracking and Two‐Point Microrheology in Cells

Mechanical stress and stiffness are increasingly recognized to play important roles in numerous cell biological processes, notably cell differentiation and tissue morphogenesis. Little definite is known, however, about how stress propagates through different cell structures or how it is converted to biochemical signals via mechanotransduction, due in large part to the difficulty of interpreting many cell mechanics experiments. A newly developed technique, two‐point microrheology (TPM), can provide highly interpretable, quantitative measurements of cells' frequency‐dependent shear moduli and spectra of their fluctuating intracellular stresses. \{TPM\} is a noninvasive method based on measuring the Brownian motion of large numbers of intracellular particles using multiple‐particle tracking. While requiring only hardware available in many cell biology laboratories, a phase microscope and digital video camera, as a statistical technique, it also requires the automated analysis of many thousands of micrographs. Here we describe in detail the algorithms and software tools used for such large‐scale multiple‐particle tracking as well as common sources of error and the microscopy methods needed to minimize them. Moreover, we describe the physical principles behind \{TPM\} and other passive microrheological methods, their limitations, and typical results for cultured epithelial cells.