Abstract
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to construct sample chambers (flow chambers) for polTIRFM motility assays. Each chamber can hold ∼20 µL of solution. To flow a solution through the chamber, the solution is added to the chamber with a pipette while wicking out the previous contents with filter paper. Each end of the coverslip should extend beyond the edge of the slide to support the pipette tip and filter paper. The flow rate can be roughly controlled by adjusting the contact area between the filter paper and the solution. The chambers can be used for investigating the motility of myosin V in vitro with the processive motility assay, as well as for assessing the motility of actin using the twirling assay.