Imaging single molecules in live cells in three dimensions.

Prof. Raimund Ober
UT Dallas and UT Southwestern Medical Center

The notions of resolution and localization have recently received attention in microscopy, especially in the context of single molecule microscopy. In this presentation we will review our work on these topics over the last several years. An important motivation and application for localization techniques is single particle tracking, in particular in three dimensions. Conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously, we had developed a novel imaging modality called multifocal plane microscopy (MUM) which enables simultaneous imaging of multiple planes within a cell-sample.
We will show that MUM is particularly well suited for the imaging of fast intracellular dynamics in three dimensions. Data will be presented to show single molecule tracking of quantum dot labeled antibody molecules over extended periods of time. We had also shown that MUM provides superior depth discrimination when capability compared to a standard microscope, which in turn paves the way for high resolution 3D single molecule tracking within a live cell environment. We also discuss a variation of the multifocal imaging approach when two opposing objectives are used. This allows for an increased amount of photons to be collected which leads to improved localization accuracy.