Quantifying cell-surface interactions using total internal reflection microscopy.

Abstract

A more thorough understanding of cell-surface interactions is needed to gain further insight into how the human body functions and to develop effective artificial cell membranes for medical use. Cellular interactions consist of specific and nonspecific forces. The specific forces, such as receptor-ligand interactions, are responsible for the actual adhesion. However, it is the nonspecific forces, such as electrostatic, van der Waals, and steric forces, that control the initial approach of a cell to a surface, which ultimately leads to adhesion. The focus of this research was to quantify the nonspecific forces acting in biological systems to facilitate a better understanding of cellular adhesion. The nonspecific forces were quantified using total internal reflection microscopy (TIRM). TIRM is a non-invasive technique that utilizes the characteristics of an evanescent wave to measure the distribution of separation distances sampled by a sphere near a flat surface. Assuming the separation distances represent a Boltzman distribution, a potential energy profile can be calculated for a single colloidal particle interacting with a flat surface. The interactions between both cells and liposomes and a glass plate were successfully measured with TIRM, although experimental challenges limited the scope of possible experiments. In response to these challenges, model cells were developed using polystyrene microspheres coated with various cell membrane components. The model cells were used to measure the effect of individual cell membrane components on the nonspecific forces. Measured potential energies of interaction between phospholipid, protein, and glycoprotein-coated spheres and a glass plate show excellent agreement with theoretical predictions based on an exponential model for the electrostatic energy. Debye lengths calculated from the experiments agree with those predicted from solution conductivity measurements. Measurements with glycolipid/phospholipid-coated spheres demonstrate that TIRM can be used to measure interactions beyond the simple sphere-plate geometry. Sources of experimental error were identified and the effects of the error on the TIRM measurements were quantified. Results show that TIRM is very effective in measuring interaction forces in model biological systems and can be used for the development of improved artificial membranes.