Going beyond 2D: Following membrane diffusion and topography in the IgE-Fc[Epsilon]RI system using 3-dimensional tracking microscopy.

The ability to follow and observe single molecules as they function in live cells represents a major milestone for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior studies of two dimensional membrane diffusion (Andrews et al., , , 955, 2008). In addition, the microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of following single molecule dynamics on live cells with three dimensional spatial resolution.