Protein transport across a cell monolayer represents a fundamental cellular process. To date the 3D spatial-temporal dynamics of such processes are poorly understood, as there is a lack of an appropriate methodology for 3D single-molecule tracking in thick samples.  We show that multifocal plane microscopy (MUM) provides a solution. While MUM was previously used for 3D single molecule tracking across shallow depths (1 micron) in live cells, the question arises if MUM can also live up to the significant challenge of tracking single molecules in a thick sample. By substantially expanding the capabilities of MUM, here we demonstrate 3D tracking of quantum-dot labeled single molecules in a ~10 micron thick live-cell monolayer, for extended periods of time (minutes). We have implemented a 4-plane MUM setup with increased focal plane spacing and a multicolor imaging approach, which enables

3D single molecule tracking in thick cellular samples using multifocal plane microscopy

242nd National Meeting (2011)

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