Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.

The imaging of cellular dynamics in three dimensions using a standard microscope is severely limited due to the fact that only one focal plane can be imaged at a given point in time. Here we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. This is achieved by a modification of the emission pathway of a standard microscope. The efficacy of the design is shown by imaging bead samples and a FcRn-GFP transfected tubule that leaves a sorting endosome and subsequently exocytoses at the plasma membrane.

The intracellular events on the recycling pathway that lead from sorting endosomes to exocytosis at the plasma membrane are central to cellular function. However, despite intensive study, these processes are poorly characterized in spatial and dynamic terms. The primary reason for this is that, to date, it has not been possible to visualize rapidly moving intracellular compartments in three dimensions in cells. Here, we use a recently developed imaging setup in which multiple planes can be simultaneously imaged within the cell in conjunction with visualization of the plasma membrane plane by using total internal reflection fluorescence microscopy. This has allowed us to track and characterize intracellular events on the recycling pathway that lead to exocytosis of the MHC Class I-related receptor, FcRn. We observe both direct delivery of tubular and vesicular transport containers (TCs) from sorting endosomes to exocytic sites at the plasma membrane, and indirect pathways in which TCs that are not in proximity to sorting endosomes undergo exocytosis. TCs can also interact with different sorting endosomes before exocytosis. Our data provide insight into the intracellular events that precede exocytic fusion.immunoglobulin G ͉ multifocal plane imaging ͉ neonatal Fc receptor ͉ sorting endosome ͉ total internal reflection