Fast 3D Single Molecule Tracking with Multifocal Plane Microscopy in Polarized Epithelia Reveals a Novel Cellular Process of Intercellular Transfer

The study of intracellular trafficking processes represents a fundamental problem in many areas of biomedical research. Single molecule imaging approaches are well suited to study heterogeneous processes in live cells. However, 3D single molecule imaging of intracellular trafficking events in a thick sample such as an epithelial-cell monolayer poses several technical challenges. Specifically, we require a methodology that not only enables fast 3D tracking of single molecules across a cell monolayer, but also enables the imaging of the cellular environment with which the single molecule interacts. Current approaches that are widely used for 3D single molecule imaging/tracking are not well suited for studying the intracellular trafficking pathways due to restricted imaging depth, poor temporal resolution, and the ability to track only a few molecules at one time. Here we show that multifocal plane microscopy (MUM), a 3D imaging modality developed by our group, provides the much needed solution to this longstanding problem by demonstrating fast 3D tracking of quantum dot labeled transferrin molecules in a ∼10 micron thick epithelial cell monolayer.The use of MUM led to the unexpected discovery of a novel cellular process, intercellular transfer, that involves the rapid exchange of Tf molecules between two adjacent cells in the monolayer. We also report 3D single molecule tracking of endocytosis and exocytosis at the lateral plasma membrane of cells in the monolayer. This lateral membrane has been notoriously difficult to image with other cellular imaging modalities. A detailed characterization of these events based on the temporal and 3D intracellular spatial behavior of Tf molecules has been made. The methods and approaches used in this study have broad applicability to investigate 3D trafficking pathways in other cell systems and models.