SUMMARY Stress granules are mRNA-protein granules that form when translation initiation is limited and are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures referred to as cores within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions, links between stress granules and human diseases, and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently; with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort we designed a competition to extensively characterise and rank the performance of 2D and 3D single molecule localization microscopy software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D, and double helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software and provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against current state of the art, and provides insight into the current limits of the field. RESULTS Competition design We established a broad committee from the SMLM community, including experimentalists and software developers, to define the scope of the challenge, ensure realism of the datasets and define analysis metrics. We opened this discussion to all interested parties in an online discussion forum 17. In 2016, we ran a first round of the 3D SMLM competition with explicit submission deadlines, culminating in a special session at the 6th annual Single Molecule Localization Microscopy Symposium (SMLMS 2016). Since then, the challenge has been opened to continuously accept new entries. Thirtysix software packages have been entered in the competition thus far, including four packages used in commercial software (Table S1, Supplementary Note 1). Participation in the competition actually led at least eight teams to modify their software to support additional 3D SMLM modalities, showing how competition can foster microscopy software development. Realistic 3D simulations Testing super-resolution software on experimental data lacks the ground truth information required for rigorous quantification of software performance. Therefore, realistic simulated datasets are required. A critical challenge to in simulating 3D SMLM data was accurate modeling of the

Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photonlimited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

The 3D orientation and location of individual molecules is an important marker for the local environment and the state of a molecule. Therefore dipole localization and orientation estimation is important for biological sensing and imaging. Precise dipole localization is also critical for superresolution imaging. We propose and analyze wide field microscope configurations to simultaneously measure these parameters for multiple fixed dipole emitters. Examination of the images of radiating dipoles reveals how information transfer and precise detection can be improved. We use an information theoretic analysis to quantify the performance limits of position and orientation estimation through comparison of the Cramer-Rao lower bounds in a photon limited environment. We show that bi-focal and double-helix polarization-sensitive systems are attractive candidates for simultaneously estimating the 3D dipole location and orientation.

36With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of 37 different data analysis packages have been developed to generate super-resolution images. To guide 38 researchers on the optimal analytical software for their experiments, we have designed, in a large 39 community effort, a competition to extensively characterise and rank these options. We generated 40 realistic simulated datasets for popular imaging modalities -2D, astigmatic 3D, biplane 3D, and double 41 helix 3D -and evaluated 36 participant packages against these data. This provides the first broad 42 assessment of 3D single molecule localization microscopy software, provides a holistic view of how 43 the latest 2D and 3D single molecule localization software perform in realistic conditions, and 44 ultimately provides insight into the current limits of the field. 45 595