Imaging of Three-Dimensional Single Molecule Dynamics in their Cellular Context

Single molecule microscopy has provided a wealth of information regarding the dynamics of receptors on the plasma membrane. In contrast, due to lack of appropriate imaging approaches, very little is known about three-dimensional (3D) single molecule trafficking in subcellular compartments. There are two major technical problems in imaging 3D subcellular single molecule dynamics. First, the dynamics of the single molecules are not confined to one focal plane of the microscope and second, having only the trajectories available of single molecules is typically not meaningful unless the cellular context of the single molecule dynamics is also imaged. For example, only by the simultaneous imaging of the single molecule dynamics and the cellular context can, for example, the diffusion of a receptor be analyzed on a non-stationary endosome. Here we introduce a novel microscopy imaging modality that is designed to simultaneously image both the dynamics of single molecules in three dimensions and the cellular context which may labeled with several fluorophores. Imaging of the single molecule dynamics is carried out using multi-focal plane microscopy (MUM), a previously demonstrated and powerful tool for this task. The cellular context is imaged using a remote focusing approach that is highly compatible with MUM imaging. The efficacy of this new imaging modality, called rMUM for remote focusing multifocal plane microscopy, is demonstrated by an investigation of the trafficking pathways of antibodies against the prostate-specific membrane antigen (PSMA) in prostate cancer cells. We demonstrate that novel experiments such as a single molecule diffusion study on sorting endosomes are possible with the new microscope, and we completely characterize the pathway of PSMA-specific antibodies, starting with diffusion towards the plasma membrane, followed by endocytosis, transport to sorting endosomes, and subsequent diffusion on sorting endosomes, and ending with either recycling or maturation into a multivesicular body.