We want to thank Tobias Pietzsch for providing his powerful open source software BigDataViewer. We thank the Light Microscopy Facility of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), namely Jan Peychl, Sebastian Bundschuh and Davide Accardi for technical assistance, perfect maintenance of the microscopes used in the study and for comments on the manuscript and H. Moon (MPI-CBG Scientific Computing Facility) for the BigDataServer. We thank Julia Eichhorn for assembling the Movie 1. The Norden lab members and Svea Grieb provided many helpful comments on the manuscript. Jaroslav Icha, Christopher Schmied and Jaydeep Sidhaye are members of the International Max Planck Research School for Cell, Developmental and Systems Biology and doctoral students at TU Dresden. Pavel Tomancak is supported by the ERC Starting Grant: Quantitative Analysis of the Hourglass Model of Evolution of Development and Human Frontier Science Program Young Investigator grant RGY0093/2012. Caren Norden is supported by the Human Frontier Science Program (CDA-00007/2011) and the German Research Foundation (DFG) [SFB 655, A25].

Open Access publication fees were provided by, Carl Zeiss Microscopy GmbH.

Morphogenesis is the process that shapes the embryo and together with growth and differentiation drives the ontogeny from a fertilized egg into a mature multicellular organism. The morphogenetic processes during animal development can be analyzed best by imaging of intact living specimens1-3. This is because such whole embryo imaging preserves all the components that drive and regulate development including gradients of signaling molecules, extracellular matrix, vasculature, innervation as well as mechanical properties of the surrounding tissue. To bridge the scales, at which morphogenesis occurs, the fast subcellular events have to be captured on a minute time scale in the context of development of the whole tissue over hours or days. To fulfill all these requirements, a modern implementation4 of the orthogonal plane illumination microscope5 was developed. Originally, it was named the Selective Plane Illumination Microscopy (SPIM)4; now an all-embracing term Light Sheet Fluorescence Microscopy (LSFM) is typically used. LSFM enables imaging at high time resolution, while inducing less phototoxicity than laser scanning or spinning disc confocal microscopes6,7. Nowadays, there are already many implementations of the basic light sheet illumination principle and it has been used to image a large variety of specimens and processes previously inaccessible to researchers8-11.
We would first like to highlight several key advantages of LSFM over conventional confocal microscopy approaches:
To acquire meaningful results from live imaging microscopic experiments, it is important that the observation only minimally affects the specimen. However, many organisms including zebrafish are very susceptible to laser light exposure, making it challenging to image them in a confocal microscope with high time resolution without phototoxicity effects like stalled or delayed development6,7. LSFM is currently the fluorescence imaging technique with the least disruptive effects on the sample7. Since the thin laser light sheet illuminates only the part of the specimen that is imaged at a particular time point, the light sheet microscope is using photons very efficiently. Consequently, the low light exposure allows for longer time-lapse observations of healthier specimens, e.g.12-17. Furthermore, thanks to the minimal invasiveness of LSFM, the number of acquired images is no longer dictated by how much light the sample can tolerate, but rather by how much data can be processed and stored.
Along the same lines of keeping the specimen in near physiological conditions, LSFM comes with alternative sample mounting strategies well suited for live embryos. In LSFM techniques, the embryos are typically embedded within a thin column of low percentage agarose. The mounting into agarose cylinders allows for the complete freedom of rotation, so the sample can be observed from the perfect angle (in LSFM referred to as view) and from several views simultaneously. The multiview imaging and subsequent multiview fusion is beneficial particularly for big, scattering specimens and allows capturing them with high, isotropic resolution. A summary of other possible LSFM mounting strategies can be found in the official microscope operating manual, in the chapter on sample preparation written by the lab of E. Reynaud. It is a recommended read, especially if the goal is to image different samples than described here.
The image acquisition in LSFM is wide field, camera-based, as opposed to laser scanning confocal microscope. This results in a higher signal-to noise ratio (SNR) for acquired images and can be extremely fast (tens to hundreds of frames per second). The high sensitivity of LSFM further enables imaging of weakly fluorescent samples, like transcription factors expressed at endogenous levels18 or, in the near future, endogenous proteins tagged using CRISPR/Cas9. The high SNR is also important for successful downstream image analysis. The high speed is required not only to capture rapid intracellular processes, but also to image the whole embryo from multiple views fast enough. A seamless fusion of the multiple views can only be achieved, if the observed phenomenon does not change during acquisition of these several z stacks coming from separate views.
The advantages of LSFM do not typically come at the expense of image quality. The lateral resolution of LSFM is slightly worse than the resolution of a confocal microscope. This is because the detection objectives used in LSFM have lower numerical aperture (usually 1.0 or less) compared to 1.2-1.3 of water or silicon immersion objectives on standard confocal setups. Additionally, due to the wide field detection in LSFM (absence of a pinhole), there is more out-of-focus light compared to a confocal microscope. The amount of out-of-focus light is determined by the light sheet thickness. Nevertheless, these disadvantages are compensated by the higher SNR in LSFM. In practice, this results in similar quality images compared to for example spinning disc confocal acquisition15. Consequently, this enables reliable extraction of features like cell membranes or nuclei, e.g., for cell lineage tracing15,19.
The axial resolution of LSFM is determined, in addition to the detection objective, by the light sheet thickness. The axial resolution of LSFM can in some cases surpass the resolution of confocal microscopes. First, the improvement in resolution comes when the light sheet is thinner than the axial resolution of the detection objective, which typically occurs for large specimens imaged with a low magnification objective. The second way, how the LSFM can achieve better axial resolution, is the multiview fusion, in which the high xy resolution information from different views is combined into one image stack. The resulting merged stack has an isotropic resolution approaching the values of resolution in the lateral direction20,21. The strategy for registering the multiple views onto each other described in this article is based on using fluorescent polystyrene beads as fiduciary markers embedded in the agarose around the sample20,21.
As a result of the LSFM commercialization, this technique is now available to a broad community of scientists22. Therefore, the motivation for writing this protocol is to make this technology accessible to developmental biologists lacking practical experience in LSFM and to get these scientists started using this technology with their samples. Our protocol uses the commercial light sheet microscope, which constitutes a conceptually simple microscope that is easy to operate. We would additionally like to mention other recent protocols for imaging zebrafish with home-built LSFM setups, which might be suitable to answer particular questions23-25. Another entry option to LSFM are the open platforms26,27, which use the open access principles to bring light sheet microscopy to a broader community. The documentation of both the hardware and the software aspects can be found at http://openspim.org and https://sites.google.com/site/openspinmicroscopy/.
In this protocol, we use the teleost zebrafish as a model system to study developmental processes with LSFM. Morphogenesis of the zebrafish eye is an example that underlines many of the benefits of LSFM. LSFM has already been used in the past to investigate eye development in medaka28 and in zebrafish29,30. At the early stage of eye development it is complicated to orient the embryo correctly for conventional microscopy, as the bulky yolk does not allow the embryo to lie on the side with its eye facing the objective. However, with LSFM mounting into an agarose column, the sample can be reproducibly positioned. Additionally, during the transition from optic vesicle to the optic cup stage, the eye undergoes major morphogenetic rearrangements accompanied by growth, which requires capturing a large z stack and a big field of view. Also for these challenges LSFM is superior to conventional confocal imaging. The process of optic cup formation is three-dimensional, therefore it is hard to comprehend and visualize solely by imaging from one view. This makes multiview imaging with isotropic resolution beneficial. After optic cup formation, the retina becomes increasingly sensitive to laser exposure. Thus, the low phototoxicity associated with LSFM is a major advantage for long-term imaging.
Here we present an optimized protocol for imaging of one to three days old zebrafish embryos and larvae with focus on the eye development. Our method allows recording of time-lapse movies covering up to 12-14 hr with high spatial and temporal resolution. Importantly, we also show a pipeline for data processing, which is an essential step in LSFM, as this technique invariably generates big datasets, often in the terabyte range.

<table><tbody><tr><td>Lightsheet Z.1 microscope</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Low melting point agarose</td><td>Roth</td><td>6351.1</td><td></td></tr><tr><td>Low melting point agarose</td><td>Sigma</td><td>A4018 or A9414</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (MESAB/MS-222/Tricaine)</td><td>Sigma</td><td>E10521</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Phenylthiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>500 nm red fluorescent beads F-Y 050</td><td>Millipore (Estapor)</td><td>80380495</td><td></td></tr><tr><td>20 &amp;mu;l (1&amp;nbsp;mm inner diameter, marked black) capilllaries</td><td>Brand</td><td>701904</td><td>sold as spare part for transferpettor</td></tr><tr><td>Teflon tip plungers for 20&amp;nbsp;&amp;mu;l capillaries</td><td>Brand</td><td>701932</td><td>sold as spare part for transferpettor</td></tr><tr><td>Circular glass coverslips diameter 18 mm, selected thickness 0.17&amp;nbsp;mm</td><td>Thermo scientific (Menzel-Glaser)</td><td></td><td></td></tr><tr><td>O-rings for chamber windows 17&amp;times;1.5 mm</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Tweezers, style 55</td><td>Dumont</td><td>0209-55-PO</td><td></td></tr><tr><td>50 ml Luer-Lock syringes</td><td>Becton Dickinson</td><td>300865</td><td></td></tr><tr><td>150 cm extension cable for infusion compatible with Luer-Lock syringes</td><td>Becton Dickinson</td><td>397400</td><td></td></tr><tr><td>&lt;strong&gt;Links&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Link 1 Multiview reconstruction application</td><td></td><td></td><td>&lt;a href=&quot;https://github.com/bigdataviewer/SPIM_Registration&quot;&gt;https://github.com/bigdataviewer/SPIM_Registration&lt;/a&gt;&lt;br /&gt;&lt;a href=&quot;http://fiji.sc/Multiview-Reconstruction&quot;&gt;http://fiji.sc/Multiview-Reconstruction&lt;/a&gt;</td></tr><tr><td>Link 2 BigDataViewer</td><td></td><td></td><td>&lt;a href=&quot;https://github.com/bigdataviewer&quot;&gt;https://github.com/bigdataviewer&lt;/a&gt;</td></tr><tr><td>Link 3 BigDataViewer</td><td></td><td></td><td>&lt;a href=&quot;http://fiji.sc/BigDataViewer&quot;&gt;http://fiji.sc/BigDataViewer&lt;/a&gt;</td></tr><tr><td>Link 4 Multiview reconstruction application-issues</td><td></td><td></td><td>&lt;a href=&quot;https://github.com/bigdataviewer/SPIM_Registration/issues&quot;&gt;https://github.com/bigdataviewer/SPIM_Registration/issues&lt;/a&gt;</td></tr><tr><td>Link 5 BigDataViewer-issues</td><td></td><td></td><td>&lt;a href=&quot;https://github.com/bigdataviewer/bigdataviewer_fiji/issues&quot;&gt;https://github.com/bigdataviewer/bigdataviewer_fiji/issues&lt;/a&gt;</td></tr><tr><td>Link 6 BigDataServer</td><td></td><td></td><td>&lt;a href=&quot;http://fiji.sc/BigDataServer&quot;&gt;http://fiji.sc/BigDataServer&lt;/a&gt;</td></tr></tbody></table>

using light sheet fluorescence microscopy to image zebrafish eye development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

LSFM is an ideal method for imaging developmental processes across scales. Several applications are compiled here showing both short and long-term imaging of intracellular structures, as well as cells and entire tissues. These examples also demonstrate that LSFM is a useful tool at various stages of eye development from optic cup formation to neurogenesis in the retina. Movie 1 serves as an illustration of the general LSFM approach, first showing a zoomed out view of an intact embryo in the embedding agarose cylinder in brightfield and later showing a detailed view of the retina in the fluorescence channel.
<img alt="Movie 1" src="/files/ftp_upload/53966/53966movie1.jpg" /> 
Movie 1: LSFM of the zebrafish retina. To illustrate the LSFM approach, this movie shows first in the brightfield that the embryo is imaged intact inside the agarose cylinder before switching to fluorescence. Later it is shown that the big field of view enables observation of the whole retina. Next, the movie shows a magnified small region of the retina to highlight the good subcellular resolution. An Ath5:gap-GFP37 transgenic zebrafish embryo was used for imaging. This transgene labels different neurons in the retina (mainly ganglion cells and photoreceptor precursors). The fluorescence part of the movie was captured as a single view recording with dual sided illumination using the 40X/1.0 W objective with 5 min intervals. The maximum intensity projection of a 30 &#181;m thick volume is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie1.mp4" target="_blank">Please click here to download this file.</a>
Movie 2 demonstrates, how very fast intracellular events can be captured with high resolution; in this case the growth of microtubules at their plus ends in the retinal neuronal progenitor cells. The information contained in the movie allows for tracking and quantification of microtubule plus end growth.
<img alt="Movie 2" src="/files/ftp_upload/53966/53966movie2.jpg" /> 
Movie 2: Microtubule dynamics in a single cell. This movie captures growing plus tips of microtubules labeled by the plus tip marker protein EB3-GFP38. The protein is expressed in a single retinal progenitor cell. The microtubules are growing predominantly in the direction from apical to basal (top to bottom). The average speed of the EB3 comets was measured as 0.28 &#177;0.05 &#181;m/sec. The bright spot at the apical side of the cell exhibiting high microtubule nucleation activity is the centrosome. The wild type embryo was injected with hsp70:EB3-GFP plasmid DNA. The movie was acquired 4 hr after heat shock (15 min at 37 &#176;C) at around 28 hrpost fertilization (hpf) as a single view recording with single sided illumination using the 63X/1.0 W objective and 1 sec time intervals. The single cell was cropped from a field of view covering the majority of the retina. The maximum intensity projection of two slices is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie2.mp4" target="_blank">Please click here to download this file.</a>
Figure 3 shows, how intracellular structures can be followed over many hours. Here, the centrosome within translocating retinal ganglion cells (RGCs) is captured.
<img alt="Figure 3" src="/files/ftp_upload/53966/53966fig3.jpg" /> 
Figure 3: Centrosome localization during the retinal ganglion cell translocation. This montage of a time-lapse experiment shows the position of the centrosome throughout the retinal ganglion cell (RGC) maturation. In the neuronal progenitors the centrosome is localized in the very tip of the apical process (1:00). During cell division, the two centrosomes serve as poles for the mitotic spindle (2:25). This division results in one daughter cell that differentiates into an RGC and a second daughter cell that becomes a photoreceptor cell precursor. After division, the cell body of the RGC translocates to the basal side of the retina, while the apical process remains attached at the apical side. Once the RGC reaches the basal side, its apical process detaches and the centrosome travels with it (6:15). The centrosome can be followed while gradually retracting together with the apical process (6:50, 7:20, 8:10). In the last frame (8:55) the ganglion cell is growing an axon from its basal side, while the centrosome is still localized apically. The mosaic expression was achieved by plasmid DNA injection into wild type embryo at one cell stage. The cells are visualized by the Ath5:GFP-caax (green) construct, which labels RGCs and other neurons. The centrosomes (arrowheads) are labeled by Centrin-tdTomato29 expression (magenta). The apical side of the retina is at the top of the image and the basal side at the bottom. The maximum intensity projection of a 30 &#181;m thick volume is shown. The images were cropped from a movie covering the whole retina. The movie starts at around 34 hr post fertilization (hpf). A z stack was acquired every 5 min with the 40X/1.0 W objective. Time is shown in hh:mm. Scale bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In Figure 4 it is displayed, how single cell behavior can be extracted from data capturing the whole tissue such as in Movie 1. Translocation of an RGC can be easily tracked and its apical and basal processes followed.
<img alt="Figure 4" src="/files/ftp_upload/53966/53966fig4.jpg" /> 
Figure 4: Translocation of a single retinal ganglion cell. The RGC translocation from apical to basal side of the retina occurs after the terminal mitosis as described in Figure 3. The RGC is labeled by expression of the Ath5:gap-GFP37 transgene. The maximum intensity projection of a 30 &#181;m thick volume is shown. The images were cropped from a movie covering the whole retina. The movie starts at around 34 hpf. A z stack was acquired every 5 min with the 40X/1.0 W objective. Time is shown in hh:mm. Scale bar represents 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 5 demonstrates the ability of multiview LSFM to capture tissue scale morphogenetic processes with cellular resolution on the example of optic cup morphogenesis, during which optic vesicle transforms into an optic cup. The image quality can be vastly improved by multiview imaging, when the final image is combined from the information out of 5 different views (in this case) into one z stack with isotropic resolution. This figure illustrates the improvement in image quality after weighted-average fusion and further gain in image contrast and resolution after multiview deconvolution. The figure shows two optical slices in different orientations through the dataset. Additionally, a montage from the deconvolved dataset shows the optic cup morphogenesis over time. All the time points are then shown in Movie 3.

<img alt="Figure 5" src="/files/ftp_upload/53966/53966fig5.jpg" /> 
Figure 5: Comparison of image quality between single view and two methods of multiview fusion. (A) One optical slice of single view data shown from lateral view and (B) dorsal view. Stripe artifacts and degradation of the signal deeper inside the sample are apparent. Also a part of the image visible in the fused data (C-F) was not captured in this particular view. (C) The same optical slice, now as multiview fused data shown from lateral view and (D) dorsal view. Note that stripe artifacts are suppressed and structures deep in the sample are better resolved. (E) The same optical slice now as multiview deconvolved data shown from lateral view and (F) dorsal view. Note the increased contrast and resolution so the individual cell membranes and nuclei can be well distinguished. The resolution does not deteriorate notably deeper inside the sample. The dataset was acquired with dual sided illumination from 5 views approximately 20 degrees apart. The z stacks of about 100 &#181;m with 1.5 &#181;m step size were acquired at each view in 10 min intervals for 10 hours with the 20X/1.0 W objective. Input images for the multiview fusion and deconvolution were down sampled 2&#215; to speed up the image processing. 15 iterations of the multiview deconvolution were run. (G) The montage shows a cropped area of the dorsal view from the deconvolved data to highlight the morphogenetic events during early eye development from the optic vesicle to the optic cup stage. The two layers of the optic vesicle, which are initially similar columnar epithelia, differentiate into distinct cell populations. The distal layer close to the epidermis becomes the retinal neuroepithelium (RN) and the proximal layer close to the neural tube becomes the retinal pigment epithelium (RP). The cells in the RN elongate and invaginate to form the optic cup (1:40 to 5:00); at the same time the RP cells flatten. The surface ectoderm is induced to form a lens (1:40), which invaginates later (5:00). The movie starts at 17 hpf. Time is shown in hh:mm. All the cellular membranes are labeled by the &#946;-actin:ras-GFP transgene and all the nuclei are labeled by the hsp70:H2B-RFP transgene. Scale bar represents 30 &#181;m. FB forebrain, LE lens, OP olfactory placode, RN retinal neuroepithelium, RP retinal pigment epithelium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 3" src="/files/ftp_upload/53966/53966movie3.jpg" /> 
Movie 3: Optic cup morphogenesis shown with single view and two methods of multiview fusion. The time-lapse movie illustrates the complete process of optic cup morphogenesis from the optic vesicle to the optic cup stage. It shows a single optical slice from the lateral view (top) and a single optical slice from the dorsal view (bottom). The cells of the developing optic vesicle undergo complex rearrangements to finally form the hemispherical optic cup with the inner retinal neuroepithelium and outer retinal pigment epithelium. A lens is formed from the surface ectoderm. It invaginates along with the neuroepithelium and sits in the optic cup. All cellular membranes are labeled by expression of the &#946;-actin:ras-GFP (green) transgene and the nuclei are labeled with hsp70:H2B-RFP (magenta). The movie starts at around 17 hpf. The dataset was acquired with dual sided illumination from 5 views approximately 20 degrees apart and a z stacks of about 100 &#181;m were acquired every 10 min with the 20X/1.0 W objective. Time is shown in hh:mm. Scale bar represents 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie3-final.mp4" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development at JoVE.com

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

using-light-sheet-fluorescence-microscopy-to-image-zebrafish-eye

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

33.8K Views.  Max Planck Institute of Molecular Cell Biology and Genetics. Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Video: Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biology

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Bioengineering

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Neuroscience

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

Light Sheet Microscopy Sample Preparation: Mounting Live Zebrafish Embryos for Long-Term Imaging

Source: Icha J., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/53966/" target="_blank">Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development</a>. J. Vis. Exp. (2016).
This article describes sample preparation for light-sheet fluorescence microscopy. It also demonstrates a method to mount live zebrafish embryos for long term imaging, which allows recording of long time-lapse movies of live embryos in near-physiological conditions.

Przygotowanie próbki do mikroskopii świetlnej: montaż żywych zarodków danio pręgowanego do długoterminowego obrazowania

Source: Icha J., et al. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development. J. Vis. Exp. (2016).
This article describes sample ...