The acquisition and analysis of polarized total internal reflection fluorescence microscopy (polTIRFM) data.

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 acquire polTIRFM data and then calibrate the setup. Calibration corrects for any systematic variations in beam intensity and unequal detector sensitivities and is performed for each slide after experimental data are recorded. To convert the intensities into angles, one set of (θ, ϕ, δ(s), δ(f), κ) is then determined from one complete cycle of the incident intensities. This process is repeated for every cycle in the trace to measure the time dependence of rotational motions. The collection and analysis of data is similar for the processive motility assay for myosin V and for the twirling filament assay, in which a sparsely labeled actin filament is translocated by a field of unlabeled myosin V.