Three-dimensional single-molecule tracking in living cells

This chapter reviews recent progress in single molecule 3D tracking in living cells. It begins with a historical overview of single particle tracking methods, moving to discussing advances necessary to move from tracking particles to molecules. We also discuss the technical means of encoding or measuring the motion of a single molecule in three spatial dimensions over time. We describe 3D single molecule tracking with both wide-field, camera-based approaches and 3D tracking based upon modified confocal microscope designs. This chapter also discusses various ways of analyzing or classifying molecular trajectories. Biophysical applications, from following protein transport in living cells to studying individual virus infection pathways, are discussed. We end with a view toward potential future improvements of 3D tracking in living cells.