Project Summary The cellular environment is both powerful and complex, depending both on structural organization from the micron scale down to the nanometer scale, as well as on the dynamic time-dependence of a huge array of enzymes, the nanomachines of the cell, and their work on proteins, oligonucleotides, and small molecules. Visible fluorescence microscopy has been a useful tool capable of non-invasively exploring cellular behavior, but the diffraction-limited resolution of conventional imaging has severely restricted the information obtainable on structures on a scale below ~200 nm. Because the primary biomolecular players in cells are in the size range on the order of 10 nm, comprehensive measurements are needed on this size scale in living systems. Super- resolution microscopy, either based on single-molecule fluorescence imaging and control of the emitting concentration, or on stimulated emission depletion, has solved this problem by enabling access to nanoscale position information down to the 10-40 nm regime and below. In addition, the complementary method of single- molecule tracking provides access to the details of motions of cellular components such as motor-driven transport or the motion of DNA or RNA. Combined with advanced three-dimensional (3D) imaging, single-particle tracking allows the full motion of specific cellular players to be observed in their actual context at high speed. It is a primary thrust of this work to develop and enhance both 3D super-resolution imaging and 3D single-particle tracking in cells by pushing the boundaries of both approaches and inventing new strategies to overcome technical limitations, which will lead to unprecedented spatial and temporal information in fixed and living cells. Research in the Moerner laboratory broadly seeks to address the limitations of super-resolution imaging and single-particle tracking in cells by physical and mathematical analysis as well as by invention of new methods. The deep motivation here is to ask the fundamental question: how can the information available from each single molecule be maximized? Two key new microscopes are under development: 3D imaging over large axial ranges using pupil plane phase modulations and a tilted light sheet, and a correlative method to use cryogenic single-molecule fluorescence localizations to annotate cryo-electron tomography reconstructions. The methodological developments of this research will be applied to a variety of critical problems in cell biology by continuing established collaborations and by developing new collaborations with well-known biologists. The bacterium, Caulobacter crescentus, remains as a useful model system for cellular development needing elucidation of the superstructures and motions of biomolecules to understand the origins of asymmetric division. The Toxoplasma gondii parasite is another fascinating organism which needs exploration with super- resolution methods. The organization of chromatin on all scales...