SHR and SCR coordinate root patterning and growth early in the cell cycle.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

VoDEx: a Python library for time annotation and management of volumetric functional imaging data.

SUMMARY: In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (e.g. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. AVAILABILITY AND IMPLEMENTATION: VoDEx is an open-source Python library and can be installed via the "pip install" command. It is released under a BSD license, and its source code is publicly accessible on GitHub (https://github.com/LemonJust/vodex). A graphical interface is available as a napari-vodex plugin, which can be installed through the napari plugins menu or using "pip install." The source code for the napari plugin is available on GitHub (https://github.com/LemonJust/napari-vodex). The software version at the time of submission is archived at Zenodo (version v1.0.18, https://zenodo.org/record/8061531).

A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy.

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color in vivo imaging. Its simple design, using readily available optical components, and its easy integration provide a low-cost solution for multi-color fluorescence imaging with increased efficiency and speed.

VoDEx: a Python library for time annotation and management of volumetric functional imaging data.

In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (eg. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. Availability and Implementation: VoDEx is an open-source Python library and can be installed via the "pip install" command. It is released under a BSD license, and its source code is publicly accessible on GitHub https://github.com/LemonJust/vodex. A graphical interface is available as a napari-vodex plugin, which can be installed through the napari plugins menu or using "pip install." The source code for the napari plugin is available on GitHub https://github.com/LemonJust/napari-vodex.

Cilia function as calcium-mediated mechanosensors that instruct left-right asymmetry.

The breaking of bilateral symmetry in most vertebrates is critically dependent upon the motile cilia of the embryonic left-right organizer (LRO), which generate a directional fluid flow; however, it remains unclear how this flow is sensed. Here, we demonstrated that immotile LRO cilia are mechanosensors for shear force using a methodological pipeline that combines optical tweezers, light sheet microscopy, and deep learning to permit in vivo analyses in zebrafish. Mechanical manipulation of immotile LRO cilia activated intraciliary calcium transients that required the cation channel Polycystin-2. Furthermore, mechanical force applied to LRO cilia was sufficient to rescue and reverse cardiac situs in zebrafish that lack motile cilia. Thus, LRO cilia are mechanosensitive cellular levers that convert biomechanical forces into calcium signals to instruct left-right asymmetry.

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture.

Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.

Quantity as a Fish Views It: Behavior and Neurobiology.

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

Regional synapse gain and loss accompany memory formation in larval zebrafish.

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670-675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604-607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull 57, 397-399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

Single-objective selective-volume illumination microscopy enables high-contrast light-field imaging.

The performance of light-field microscopy is improved by selectively illuminating the relevant subvolume of the specimen with a second objective lens. Here we advance this approach to a single-objective geometry, using an oblique one-photon illumination path or two-photon illumination to accomplish selective-volume excitation. The elimination of the second orthogonally oriented objective to selectively excite the volume of interest simplifies specimen mounting; yet, this single-objective approach still reduces the out-of-volume background, resulting in improvements in image contrast, effective resolution, and volume reconstruction quality. We validate our new, to the best of our knowledge, approach through imaging live developing zebrafish, demonstrating the technology's ability to capture imaging data from large volumes synchronously with high contrast while remaining compatible with standard microscope sample mounting.

A versatile, multi-laser twin-microscope system for light-sheet imaging.

Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright (with provision for inverted) detection geometry. We offer a detailed description of the twin-microscope design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument's versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

Genetic and neuronal regulation of sleep by neuropeptide VF.

Sleep is an essential and phylogenetically conserved behavioral state, but it remains unclear to what extent genes identified in invertebrates also regulate vertebrate sleep. RFamide-related neuropeptides have been shown to promote invertebrate sleep, and here we report that the vertebrate hypothalamic RFamide neuropeptide VF (NPVF) regulates sleep in the zebrafish, a diurnal vertebrate. We found that NPVF signaling and npvf-expressing neurons are both necessary and sufficient to promote sleep, that mature peptides derived from the NPVF preproprotein promote sleep in a synergistic manner, and that stimulation of npvf-expressing neurons induces neuronal activity levels consistent with normal sleep. These results identify NPVF signaling and npvf-expressing neurons as a novel vertebrate sleep-promoting system and suggest that RFamide neuropeptides participate in an ancient and central aspect of sleep control.

SERCA directs cell migration and branching across species and germ layers.

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration.

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines.

In vivo single-cell labeling by confined primed conversion.

Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.

Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Dorsal-ventral gene expression in the Drosophila embryo reflects the dynamics and precision of the dorsal nuclear gradient.

Patterning of the dorsal-ventral axis in the early Drosophila embryo depends on the nuclear distribution of the Dorsal transcription factor. Using live two-photon light-sheet microscopy, we quantified the nuclear Dorsal gradient in space and time and found that its amplitude and basal levels display oscillations throughout early embryonic development. These dynamics raise questions regarding how cells can reproducibly establish patterns of gene expression from a rapidly varying signal. We therefore quantified domains of Dorsal target genes, discovering their expression patterns are also dynamic. Computational modeling of this system reveals a correlation between Dorsal gradient dynamics and changes in target gene expression and suggests that these dynamics, together with time averaging of noise, results in the formation of graded gene expression borders in regions where the gradient is nearly flat. We propose that mRNA levels remain plastic during transient signaling events, allowing tissues to refine patterns in the face of genetic or environmental variation.

Advances in multiphoton microscopy for imaging embryos.

Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology.