Recording light microscopic images of large, non-transparent specimens, such as developing multi-cellular organisms, is complicated by decreased contrast due to light scattering. Early zebrafish development can be captured by standard light sheet microscopy; however, new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM) with incoherent structured illumination microscopy and created structured illumination patterns with continuously adjustable frequencies (DSLM-SI). Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 hours and fast multiple-view imaging of early Drosophila development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a Fly Digital Embryo.

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.fast 4D imaging | axon growth | neuron migration P roper neural circuit assembly requires the coordinated execution of multiple events, including cell migration, axon guidance, and synaptogenesis (1, 2). During neurodevelopment, these events are orchestrated between pre-and postsynaptic partners, resulting in the correct wiring of the nervous system. The mechanisms that enable proper wiring in vivo are not well understood.Caenorhabditis elegans provides an excellent model to understand how neural circuit assembly occurs in vivo. With only 302 neurons, ∼7,000 synapses, and an available and comprehensive neural connectivity map (3), the nervous system of C. elegans is well characterized and relatively simple. The molecular mechanisms that control neurodevelopmental decisions in the nematode are well conserved throughout evolution (4), and several genetic programs that control terminal differentiation of neuronal identity were first identified and characterized in C. elegans (5, 6). Studies in C. elegans have also significantly contributed to our understanding of neuroblast migration and axon guidance (7,8).

We present an interactive method for cropping photographs given minimal information about the location of important content, provided by eye tracking. Cropping is formulated in a general optimization framework that facilitates adding new composition rules, as well as adapting the system to particular applications. Our system uses fixation data to identify important content and compute the best crop for any given aspect ratio or size, enabling applications such as automatic snapshot recomposition, adaptive documents, and thumbnailing. We validate our approach with studies in which users compare our crops to ones produced by hand and by a completely automatic approach. Experiments show that viewers prefer our gaze-based crops to uncropped images and fully automatic crops.

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