Three-dimensional single molecule microscopy enables the study of dynamic processes in living cells at the level of individual molecules. Multifocal plane microscopy (MUM) is an example of such a modality and has been shown to be capable of capturing the rapid subcellular trafficking of single molecules in thick samples by simultaneously imaging distinct focal planes within the sample. Regardless of the specific modality, however, the obtained 3D trajectories of single molecules often do not fully reveal the biological significance of the observed dynamics. This is because the missing cellular context is often also needed in order to properly understand the events observed at the molecular level. We introduce the remote focusing-MUM (rMUM) modality, which enables 3D single molecule imaging with the simultaneous z-stack imaging of the surrounding cellular structures. Using rMUM, we demonstrate the 3D tracking of prostate-specific membrane antigen (PSMA) with a PSMA-specific antibody in a prostate cancer cell. PSMA is an important biomarker for prostate cancer cells. As such, it is a common target for antibody-based therapies. For example, of particular interest is the use of PSMA-specific antibodies that are conjugated with a toxin that kills prostate cancer cells. We analyze here the pathways of PSMA-specific antibodies, from prior to their first binding to PSMA at the plasma membrane to their arrival at, and continued movement in, sorting endosomes. By making possible the observation of single molecule dynamics within the relevant cellular context, rMUM allows, in our current application, the identification and analysis of different stages of the PSMA-specific antibody trafficking pathway
