We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

We consider the problem of optimizing general convex objective functions with nonnegativity constraints. Using the Karush-Kuhn-Tucker (KKT) conditions for the nonnegativity constraints we will derive fast multiplicative update rules for several problems of interest in signal processing, including non-negative deconvolution, point-process smoothing, ML estimation for Poisson Observations, nonnegative least squares and nonnegative matrix factorization (NMF). Our algorithm can also account for temporal and spatial structure and regularization. We will analyze the performance of our algorithm on simultaneously recorded neuronal calcium imaging and electrophysiology data.

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps.

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc.

Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head or their body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye. Here we show that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. We show that when the retina moves, visual receptive fields shift accordingly, and that even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, we find that Drosophila perform binocular, vergence movements of their retinas-which could enhance depth perception-when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution.

Muscle strength testing is routine in clinical practice. Here we provide an aid to the documentation and visual conceptualization of those results - MuscleViz: a free, open-source application for visualizing the results of muscle strength testing. Its use in clinical settings streamlines the communication of physical examination findings. The tool is also useful for presenting patient data in case reports or case series. A push towards free, open-source software has benefitted other areas of science; we believe a similar effort dedicated to the development of clinical tools is worth pursuing.

Aversive olfactory memory is formed in the mushroom bodies in Drosophila melanogaster. Memory retrieval requires mushroom body output, but the manner in which a memory trace in the mushroom body drives conditioned avoidance of a learned odor remains unknown. To identify neurons that are involved in olfactory memory retrieval, we performed an anatomical and functional screen of defined sets of mushroom body output neurons. We found that MB-V2 neurons were essential for retrieval of both short- and long-lasting memory, but not for memory formation or memory consolidation. MB-V2 neurons are cholinergic efferent neurons that project from the mushroom body vertical lobes to the middle superiormedial protocerebrum and the lateral horn. Notably, the odor response of MB-V2 neurons was modified after conditioning. As the lateral horn has been implicated in innate responses to repellent odorants, we propose that MB-V2 neurons recruit the olfactory pathway involved in innate odor avoidance during memory retrieval.