3D single molecule tracking in thick cellular specimens with multifocal plane microscopy

Intracellular Protein trafficking represents a fundamental process that is important for the normal functioning of the cell. The study of protein transport merits the use of single molecule imaging approaches, as it enables the analysis of the individual transport pathways that is not feasible through conventional imaging techniques. However, 3D single molecule tracking poses several challenges, especially in thick samples such as a 10 μm thick cell monolayer. Current approaches to 3D single molecule tracking are not well suited for imaging the trafficking pathways in a cell monolayer due to technical limitations such as restricted imaging depth, poor temporal resolution, and the ability to track only a few molecules at one time.