Magnetic resonance imaging (MRI) has transformed our understanding of the human brain through well-replicated mapping of abilities to specific structures (for example, lesion studies) and functions1–3 (for example, task functional MRI (fMRI)). Mental health research and care have yet to realize similar advances from MRI. A primary challenge has been replicating associations between inter-individual differences in brain structure or function and complex cognitive or mental health phenotypes (brain-wide association studies (BWAS)). Such BWAS have typically relied on sample sizes appropriate for classical brain mapping4 (the median neuroimaging study sample size is about 25), but potentially too small for capturing reproducible brain–behavioural phenotype associations5,6. Here we used three of the largest neuroimaging datasets currently available—with a total sample size of around 50,000 individuals—to quantify BWAS effect sizes and reproducibility as a function of sample size. BWAS associations were smaller than previously thought, resulting in statistically underpowered studies, inflated effect sizes and replication failures at typical sample sizes. As sample sizes grew into the thousands, replication rates began to improve and effect size inflation decreased. More robust BWAS effects were detected for functional MRI (versus structural), cognitive tests (versus mental health questionnaires) and multivariate methods (versus univariate). Smaller than expected brain–phenotype associations and variability across population subsamples can explain widespread BWAS replication failures. In contrast to non-BWAS approaches with larger effects (for example, lesions, interventions and within-person), BWAS reproducibility requires samples with thousands of individuals.

the quality of super-resolution images obtained by singlemolecule localization microscopy (smlm) depends largely on the software used to detect and accurately localize point sources. in this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. these metrics reflect the various tradeoffs of smlm software packages and help users to choose the software that fits their needs.We have conducted a large-scale comparative study of software packages developed in the context of SMLM, including recently developed algorithms. We designed realistic data that are generic and cover a broad range of experimental conditions and compared the software packages using a multiple-criterion quantitative assessment that is based on a known ground truth.Our study is based on the active participation of developers of SMLM software. More than 30 groups have participated so far, and the study is still under way. We provide participants access to our benchmark data as an ongoing public challenge. Participants run their own software on our data and report their list of localized particles for evaluation. The results of the challenge are accessible online and updated regularly.SMLM was demonstrated in 2006, independently by three research groups 1-3 , and has enabled subsequent breakthroughs in diverse fields 4,5 . SMLM can resolve biological structures at the nanometer scale (typically 20 nm lateral resolution), circumventing Abbe's diffraction limit. At the cost of a relatively simple setup 6,7 , it has opened exciting new opportunities in life science research 8,9 .The underlying principle of SMLM is the sequential imaging of sparse subsets of fluorophores distributed over thousands of frames, to populate a high-density map of fluorophore positions. Such large data sets require automated image-analysis algorithms to detect and precisely infer the position of individual fluorophore, taking advantage of their separation in space and time.The acquired data cannot be visualized directly; further computerized image-reconstruction methods are required. These typically comprise four steps: preprocessing, detection, localization and rendering. Preprocessing reduces the effects of the background and noise; detection identifies potential molecule candidates in each frame; localization performs a subpixel refine...

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

Summary Skeletal muscle is a complex tissue containing tissue resident muscle stem cells (satellite cells, MuSCs) important for postnatal muscle growth and regeneration. Quantitative analysis, biological function, and the molecular pathways responsible for a potential juxtavascular niche for MuSCs is currently lacking. We utilized fluorescent reporter mice and muscle tissue clearing to investigate the proximity of MuSCs to capillaries in 3-dimensions. We show that MuSCs express abundant VEGFA, which recruits endothelial cells (ECs) in vitro, whereas both blocking VEGFA by a VEGF inhibitor and MuSC-specific VEGFA gene deletion reduce the proximity of MuSCs to capillaries. Importantly, this proximity to the blood vessels was associated with MuSC self-renewal in which EC-derived Notch ligand Dll4 induces quiescence in MuSCs. We hypothesize that MuSCs recruit capillary ECs via VEGFA, and in return ECs maintain MuSC quiescence though Dll4.

We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.bacterial cytoskeleton | SOS response

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.

Gene editing with CRISPR/Cas9 is revolutionizing biotechnology and medical research, yet affordable, efficient, and tailorable delivery systems are urgently needed to advance translation. Herein, a series of monodisperse amphiphilic block polymers poly­[ethylene oxide-b-2-(dimethylamino) ethyl methacrylate-b-n-butyl methacrylate] (PEO-b-PDMAEMA-b-PnBMA) that housed three PEO lengths (2, 5, and 10 kDa) and a variant lacking PEO (PDMAEMA-b-PnBMA) were synthesized via controlled radical polymerization and assembled into well-defined spherical cationic micelles. The cationic micelles were complexed via electrostatic interactions with Cas9 protein/guide RNA ribonucleoproteins (RNPs) that exhibit anionic charges due to the overhanging RNA. The resulting micelleplex formulations in both phosphate-buffered saline (PBS) and water were screened via high content analysis for gene editing efficiency. The micelle variant with the 10 kDa PEO block offered the highest gene editing performance and was advanced for in-depth characterization. For the first time, quantitative static and dynamic light scattering characterization and cryogenic transmission electron microscopy images of Cas9 protein/guideRNA RNP loading into well-defined micelleplex nanoparticles are revealed, where the formulation solvent was found to play a major role in the physicochemical properties and biological performance. In PBS, the solutions containing the micelles (63 triblock polymers per micelle) were assembled with the Cas9 protein/guideRNA RNP payloads offering uniform loading of 14 RNPs per micelleplex and moderate editing efficiency; this homogeneous system offers promise for future in vivo/preclinical applications. Interestingly, when the uniform micelles were formulated with the RNP payloads in water, larger multimicelleplex nanoparticles were formed that offered double the editing efficiency of Lipofectamine 2000 (40% gene editing) due to the rapid sedimentation kinetics of the larger colloids onto adherent cells, offering promising in vitro, ex vivo, and/or cell therapy applications. This work presents the first quantitative demonstration of tailorable block polymer micelle formulations for advancing CRISPR/Cas9 RNP delivery and fundamental correlation of the solutions physics to biological performance.

SUMMARY Salmonella enterica (Se) bacteria cause persistent intracellular infections while stimulating a robust interferon-γ-producing CD4+ T (Th1) cell response. We addressed this paradox of concomitant infection and immunity by tracking fluorescent Se organisms in mice. Se bacteria persisted in nitric oxide synthase (iNOS)-producing resident and recruited macrophages while inducing genes related to protection from nitric oxide (NO). Se-infected cells occupied iNOS+ splenic granulomas that excluded T cells but were surrounded by mononuclear phagocytes producing the chemokines CXCL9 and CXCL10, and Se epitope-specific Th1 cells expressing CXCR3, the receptor for these chemokines. Blockade of CXCR3 inhibited Th1 occupancy of CXCL9/10-dense regions, reduced activation of the Th1 cells, and lead to increased Se growth. Thus, intracellular Se bacteria survive in their hosts by counteracting toxic products of the innate immune response and by residing in T cell-sparse granulomas, away from abundant Th1 cells positioned via CXCR3 in a bordering region that act to limit infection.