We present a method to measure the molecular orientation and rotational mobility of single-molecule emitters by designing and implementing a Tri-spot point spread function. It can measure all degrees of freedom related to molecular orientation and rotational mobility. Its design is optimized by maximizing the theoretical limit of its measurement precision. We evaluate the precision and accuracy of the Tri-spot PSF by measuring the orientation and effective rotational mobility of single fluorescent molecules embedded in a polymer matrix.

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Oumeng Zhang, Tianben Ding, Jin Lu, Hesam Mazidi, and Matthew D. Lew, "Measuring 3D molecular orientation and rotational mobility using a Tri-spot point spread function," Proc. SPIE 10500, Single Molecule Spectroscopy and Superresolution Imaging XI, 105000B (Presented at SPIE BiOS: January 27, 2018; Published: 20 February 2018); https://doi.org/10.1117/12.2287004.