Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

Nervous systems have evolved to combine environmental information with internal state to select and generate adaptive behavioral sequences. To better understand these computations and their implementation in neural circuits, natural behavior must be carefully measured and quantified. Here, we collect high spatial resolution video of single zebrafish larvae swimming in a naturalistic environment and develop models of their action selection across exploration and hunting. Zebrafish larvae swim in punctuated bouts separated by longer periods of rest called interbout intervals. We take advantage of this structure by categorizing bouts into discrete types and representing their behavior as labeled sequences of bout types emitted over time. We then construct probabilistic models—specifically, marked renewal processes—to evaluate how bout types and interbout intervals are selected by the fish as a function of its internal hunger state, behavioral history, and the locations and properties of nearby prey. Finally, we evaluate the models by their predictive likelihood and their ability to generate realistic trajectories of virtual fish swimming through simulated environments. Our simulations capture multiple timescales of structure in larval zebrafish behavior and expose many ways in which hunger state influences their action selection to promote food seeking during hunger and safety during satiety.

Social isolation strongly modulates behavior across the animal kingdom. We utilized the fruit fly to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide (), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of neurons each similarly increased isolation-driven aggression. Our results suggest a U-shaped dependence of social isolation-induced aggressive behavior on signaling, similar to the actions of many neuromodulators in other contexts.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with <10 nm isotropic resolution of each section, and these can then be stitched together to span the sectioned volume. Incorporating gas cluster ion beam SEM into existing single-beam and multibeam SEM workflows should be straightforward, increasing reliability while improving z resolution by a factor of three or more.