OSA/SPIE Seminar: Computational Optics for Multidimensional Nanoscale Imaging of Single Fluorescent Molecules
Visualizing the dynamic movements and interactions between biomolecules remains a challenge, motivating the development of new optical technology and computational algorithms for imaging at the nanoscale. We have built two technologies for multidimensional imaging of single molecules (SMs): the Tri-spot point spread function (PSF) and the Robust Statistical Estimation (RoSE) algorithm. The Tri-spot PSF measures each second moment of SM orientation with near-uniform sensitivity, thereby capturing the orientation and rotational diffusion of SMs using just one camera frame. For 3D imaging, we developed RoSE to minimize the vectorial localization errors in super-resolution microscopy that result from both the structure of the sample and the PSF itself. By estimating the likelihood of a blinking event to be present in each imaging frame, RoSE localizes molecules accurately and minimizes false localizations even when images overlap.