Simultaneous Position and Orientation Imaging of Polarized Fluorescence from Rod-In-Rod Semiconductor Nanoparticles on Cytoplasmic Dynein

Abstract

Sub-pixel particle tracking and polarized total internal reflection fluorescence (polTIRF) microscopy have been instrumental in understanding of motor protein function. We have developed a method combining sub-pixel tracking and polTIRF microscopy to simultaneously track position and orientation of single fluorescent particles. The sample is illuminated with circularly polarized light, and the emission is split into four polarizations and imaged with an EMCCD camera. The Yanagida lab recently published such a technique (Ohmachi et al, PNAS vol. 109, 2012). Our lab has developed a similar method, incorporating calibration routines to compensate for depolarization by phase shifts at reflecting surfaces and wavelength- and polarization-dependent detection efficiencies of the camera channels. The application of a 4x4 calibration matrix, generated by measuring deviations from theoretical polarized fluorescence intensities, compensates for depolarization caused by reflecting surfaces and channel crosstalk. Normalizing to an unpolarized sample at the relevant emission wavelength accounts for wavelength-dependent differential sensitivity of the four detection channels. Rod-in-rod CdSe core, CdS shell particles exhibit high polarization ratios; rods with dimensions of only ∼5x20 nm display an average polarization ratio of 0.81. This position and orientation tracking method is currently being used to observe rotations of streptavidin-conjugated quantum rods bound to the cytoplasmic dynein AAA ring. Correlation of angular and translocation information may provide insight into mechanisms of fluctuating dynein stepping. Supported by NIH grant P01GM087253.