Abstract: Biomolecules, including DNA, RNA, and proteins, are the machinery critical for keeping cells healthy. However, visualizing the dynamic movements and interactions between biomolecules remains a challenge, preventing scientists from getting a clear picture of how molecular structure impacts health and disease. This challenge motivates my lab to invent optical technology and image processing algorithms for imaging at the nanoscale. We have built the Tri-spot point spread function (PSF) for imaging the orientation and rotational mobility of single molecules. The Tri-spot PSF can measure the orientations of many molecules simultaneously using one camera frame, resolve orientational dynamics that cannot be seen by other methods, and has optimized sensitivity towards all types of rotational motion. We have also developed the Robust Statistical Estimation (RoSE) algorithm for minimizing the structural bias in super-resolution microscopy. RoSE works for any 3D microscope and uses spatial and temporal sparsity to calculate the statistical confidence that localized molecule in raw image data truly represents the biological structure of interest.