Black Dots: Reference-Free Traction Force Microscopy for Measuring Single-Cell Forces

Abstract

Cells generate cytoskeletal forces for a many purposes including cell locomotion and tissue functions like heart beating and wound healing. The amount of force produced by a cell depends on many things including its cell type, size, and environment. Measuring the forces produced by cells can provide insight into their behavior and physiological function, or can be used to study the effect of drugs and disease. To isolate their behavior, forces are measured in single cells, typically on a 2D surface for simplicity, yielding "traction forces" as a cell pulls parallel to the 2D plane. This principle has been used to develop techniques such as membrane wrinkling, microposts, and traction force microscopy among many others. However, existing traction force methods have several drawbacks including the limited number of cells that can be measured per experiment or inadvertent impact on cell functions by strictly constraining cell size and shape. In this work, a novel reference-free traction force microscopy technique is developed to overcome limitations from existing methods. The technique, named "black dots", microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. Chapter 2 describes the theory and development behind the black dots approach. Several techniques are combined including microcontact printing and sacrificial films to deposit a fluorescent pattern on very soft PDMS with high fidelity. The pattern is characterized and an example data set is generated to study the sensitivity of the black dots approach. In Chapter 3 the technique is demonstrated by assessing forces in human platelets, which are the smallest cells in the human body and can generate very large forces for their size. Because the black dots approach is reference-free, fixation and immunofluorescent staining can be applied along with geometric measurements to accompany the traction force data. In this study, platelets that exert more force tend to have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by the black dots approach, a clustering analysis and a multivariate mixed effects model with interaction terms can be used to identify clusters and trends between force, area, circularity, and F-actin dispersion. Finally, Chapter 4 explores the use of black dots in measuring forces in live, dynamically beating cardiomyocytes, including a protocol and special considerations for using black dots with cardiomyocytes to ultimately measure both isometric and twitch forces over time. These forces as well as the twitch dynamics are analyzed for a small set of example cells that showcase the potential for new discoveries enabled black dots.