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Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular traffcking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insuffcient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rMUM), as it extends multifocal plane microscopy (MUM) with a remote focusing module. MUM is a modality that uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 3D single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z-stack of the specimen without having to move the microscope's objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM's remote focusing module thus allows the cellular context to be imaged in the form of z-stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context