Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

The GFP reconstitution across synaptic partners (GRASP) technique, based on functional complementation between two nonfluorescent GFP fragments, can be used to detect the location of synapses quickly, accurately and with high spatial resolution. The method has been previously applied in the nematode and the fruit fly but requires substantial modification for use in the mammalian brain. We developed mammalian GRASP (mGRASP) by optimizing transmembrane split-GFP carriers for mammalian synapses. Using in silico protein design, we engineered chimeric synaptic mGRASP fragments that were efficiently delivered to synaptic locations and reconstituted GFP fluorescence in vivo. Furthermore, by integrating molecular and cellular approaches with a computational strategy for the three-dimensional reconstruction of neurons, we applied mGRASP to both long-range circuits and local microcircuits in the mouse hippocampus and thalamocortical regions, analyzing synaptic distribution in single neurons and in dendritic compartments.

Animals learn both whether and when a reward will occur. Neural models of timing posit that animals learn the mean time until reward perturbed by a fixed relative uncertainty. Nonetheless, animals can learn to perform actions for reward even in highly variable natural environments. Optimal inference in the presence of variable information requires probabilistic models, yet it is unclear whether animals can infer such models for reward timing. Here, we develop a behavioral paradigm in which optimal performance required knowledge of the distribution from which reward delays were chosen. We found that mice were able to accurately adjust their behavior to the SD of the reward delay distribution. Importantly, mice were able to flexibly adjust the amount of prior information used for inference according to the moment-by-moment demands of the task. The ability to infer probabilistic models for timing may allow mice to adapt to complex and dynamic natural environments.

Michael Akam has been awarded the 2007 Kowalevsky medal for his many research accomplishments in the area of evolutionary developmental biology. We highlight three tributaries of Michaels contribution to evolutionary developmental biology. First, he has made major contributions to our understanding of development of the fruit fly, Drosophila melanogaster. Second, he has maintained a consistent focus on several key problems in evolutionary developmental biology, including the evolving role of Hox genes in arthropods and, more recently, the evolution of segmentation mechanisms. Third, Michael has written a series of influential reviews that have integrated progress in developmental biology into an evolutionary perspective. Michael has also made a large impact on the field through his effective mentorship style, his selfless promotion of younger colleagues, and his leadership of the University Museum of Zoology at Cambridge and the European community of evolutionary developmental biologists.

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

While the field of synthetic developmental biology has traditionally focused on the study of the rich developmental processes seen in metazoan systems, an attractive alternate source of inspiration comes from microbial developmental models. Microbes face unique lifestyle challenges when forming emergent multicellular collectives. As a result, the solutions they employ can inspire the design of novel multicellular systems. In this review, we dissect the strategies employed in multicellular development by two model microbial systems: the cellular slime mold Dictyostelium discoideum and the biofilm-forming bacterium Bacillus subtilis. Both microbes face similar challenges but often have different solutions, both from metazoan systems and from each other, to create emergent multicellularity. These challenges include assembling and sustaining a critical mass of participating individuals to support development, regulating entry into development, and assigning cell fates. The mechanisms these microbial systems exploit to robustly coordinate development under a wide range of conditions offer inspiration for a new toolbox of solutions to the synthetic development community. Additionally, recreating these phenomena synthetically offers a pathway to understanding the key principles underlying how these behaviors are be coordinated naturally.

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.