Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.

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Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.

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In recent years, the application of high-performance and distributed computing in scientific practice has become increasingly wide-spread. Among the most widely available platforms to scientists are clusters, grids, and cloud systems. Such infrastructures currently are undergoing revolutionary change due to the integration of many-core technologies, providing orders-of-magnitude speed improvements for selected compute kernels. With high-performance and distributed computing systems thus becoming more heterogeneous and hierarchical, programming complexity is vastly increased. Further complexities arise because urgent desire for scalability, and issues including data distribution, software heterogeneity, and ad-hoc hardware availability, commonly force scientists into simultaneous use of multiple platforms (e.g. clusters, grids, and clouds used concurrently). A true computing jungle. In this chapter we explore the possibilities of enabling efficient and transparent use of Jungle Computing Systems in every-day scientific practice. To this end, we discuss the fundamental methodologies required for defining programming models that are tailored to the specific needs of scientific researchers. Importantly, we claim that many of these fundamental methodologies already exist today, as integrated in our Ibis high-performance distributed programming system. We also make a case for the urgent need for easy and efficient Jungle Computing in scientific practice, by exploring a set of state-of-the-art application domains. For one of these domains we present results obtained with Ibis on a real-world Jungle Computing System. The chapter concludes by exploring fundamental research questions to be investigated in the years to come.