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Girstmair et al. BMC Developmental Biology (2016) 16:22 DOI 10.1186/s12861-016-0122-0 METHODOLOGY ARTICLE Open Access Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development Johannes Girstmair1, Anne Zakrzewski1, François Lapraz1,2, Mette Handberg-Thorsager3, Pavel Tomancak3, Peter Gabriel Pitrone3, Fraser Simpson1 and Maximilian J. Telford1* Abstract Background: Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory’s own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri – a non-model animal. Results: Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. Conclusions: We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions. * Correspondence: m.telford@ucl.ac.uk 1 Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Girstmair et al. BMC Developmental Biology (2016) 16:22 Background Light-sheet illumination for microscopy is an old technology enjoying a dramatic recent renaissance due to introduction of selective plane illumination microscopy (SPIM) [1]. The principle of SPIM is to use optics to form a thin sheet of light that passes through the specimen. Unlike a standard microscope in SPIM the objective lens is placed perpendicular to the direction of the light such that the sheet of light illuminates the specimen only at the focal plane of the lens. This has two important benefits; it eliminates scattered light from out of focus areas of the specimen providing a natural means of optical sectioning and, because only the imaged area is illuminated, the total amount of light hitting the specimen is orders of magnitude less than in conventional fluorescence microscopy meaning that photodamage/phototoxicity is enormously reduced and imaging over long periods is possible [1]. This latter benefit is of great significance for live imaging. OpenSPIM is an open access light-sheet microscopy design [2]; http:// openspim.org; see also [3]. The OpenSPIM resource gives users step-by-step guidance for building a basic configuration of a SPIM microscope and includes appropriate Page 2 of 16 open source software for image acquisition and processing such as Fiji (http://fiji.sc/Fiji), micromanager (https:// www.micro-manager.org/), multiview reconstruction plugins [4, 5] deconvolution [6] and big data viewer (http:// fiji.sc/BigDataViewer). The design can be adapted and upgraded according to the users specific requirements and budget. We have designed an OpenSPIM microscope capable of dual-sided illumination (the so called Tconfiguration proposed on the OpenSPIM wiki). The microscope was built following instructions from the website http://openspim.org with modifications required to extend the capabilities of the basic single sided illumination described there (Fig. 1). To test our system we have imaged the early embryogenesis and the larval stage of the polyclad flatworm Maritigrella crozieri, a promising new evo-devo model within Plathyhelminthes [7]. Eggs of this polyclad flatworm undergo stereotypical spiral cleavage to produce a ciliated planktotrophic larval stage known as Müller larva that shows morphological similarities to planktotrophic larval stages found in marine annelids and molluscs. The embryonic and post-embryonic development Fig. 1 OpenSPIM with dual-sided illumination, hardware-controlled laser triggering and all hardware components Girstmair et al. BMC Developmental Biology (2016) 16:22 of M. crozieri has been previously described [8]. Recent flatworm phylogenies confirm the basal position of polyclad flatworms within the rhabditophoran Platyhelminthes [9, 10] making M. crozieri and other polyclad flatworms an interesting system for evo-devo studies within Platyhelminthes and amongst other Lophotrochozoa. Here we demonstrate that, on both live and fixed material, we were able to visualize the stereotypical spiral cleavage pattern of M. crozieri with high-speed time-lapse sequences and were able to 3D-reconstruct a number of individual time points of the early embryonic development using Fiji’s bead based registration software and multi-view deconvolution plugins [4, 6]. In this report we describe a real life experience of building an OpenSPIM microscope. We discuss the difficulties we encountered, the real costs involved including the time spent and difficulties encountered as well as describing the limitations and significant benefits of the system. Results Our OpenSPIM produces high quality images which we compare to scanning electron micrographs (SEM) To test whether our OpenSPIM microscope can produce high quality images, we stained fixed 1 day old Müller’s larvae with a monoclonal Mouse anti-Acetylated Tubulin Page 3 of 16 antibody (Sigma) and used a secondary antibody conjugated to Alexa Fluor® 568 Goat anti-Mouse (Invitrogen™). Our OpenSPIM images (shown as maximum projections) show cilia covering the whole epidermis of the polyclad larva (Fig. 2a-e). This dense film of short cilia can easily be distinguished from longer cilia comprising the ciliary band along the eight lobes (Fig. 2e). Our OpenSPIM images show a clear resemblance to scanning electron microscopy images [7] of similar stage larvae (Fig. 2d, e and f) confirming reliable image acquisition with OpenSPIM at the level of embryo morphology. For a more detailed comparison of several views see also Additional file 1. The advantage of OpenSPIM multi-view reconstructions over confocal microscopy and single image in M. crozieri Standard confocal microscopes lack the possibility of multi-view imaging and reconstruction. This is important for the study of M. crozieri larvae and embryos due to the attenuation of light intensity caused by the opaque yolk meaning we can only visualize one side of the embryo. We have found that the opacity causes significant signal loss, which becomes especially obvious in Maritigrella when the confocal z-stack of an imaged specimen is rotated. We demonstrate this here in a fixed Fig. 2 Maximum projections of fixed Müller’s larvae stained with Acetylated tubulin and imaged with OpenSPIM (a-e). a Anterior view b Lateral view c Ventral view d Posterior view e magnified view of area boxed in d (f) Posterior view of a M. crozieri larval stage obtained with scanning electron microscopy for comparison with imaging acquired by our OpenSPIM. Ap, apical plate; oh, oral hood; vll, ventro-lateral lobe; ll, lateral lobe; dll, dorso-lateral lobe; sop, sub-oral plate; cb, ciliary bands (long cilia). All scalebars are 50 μm Girstmair et al. BMC Developmental Biology (2016) 16:22 larva labeled with the nucleic-stain SytoxGreen (Fig. 3, right). The second drawback of a confocal is the necessity of using a slide with coverslip, which tends to cause deformation of our topologically complex larvae. The rotation of a confocal imaged larva reveals the slightly squeezed body shape of the larva. Confocal z-stacks are not suitable for further image processing (e.g. image pattern registration as described by [11, 12]. Page 4 of 16 In contrast to confocal imaging, OpenSPIM offers a multi-view reconstruction method, whereby different angles of the same specimen can be fused into a single zstack as shown in Fig. 3 (left side). The reconstructed larva not only keeps its natural shape, but also includes the information obtained from each individual angle, resulting in a whole-mount containing high-resolution signal from all sides. For Maritigrella larvae and embryos multi-view reconstructions achieved with Fig. 3 A comparison of a multi-view reconstructed larva (multi-view deconvolution of several angles) stained with the nucleic marker SytoxGreen (left side) with a larva with the same staining captured with a Leica TCS SP8 confocal laser microscopy (right side) Girstmair et al. BMC Developmental Biology (2016) 16:22 OpenSPIM create a crucial advantage over confocal microscopy. We further visualized to what extent average fusion and deconvolution [6] can improve results over a single view in an M. crozieri embryo when imaged with our OpenSPIM. This is relevant e.g. for early cleavage observations, when nuclei of the macromeres shift from the animal pole towards the vegetal pole of the embryo and are thus difficult to see. In terms of imaging the entire embryo, it is not surprising that we found a clear benefit gained by applying multi-view imaging (average fusion or multi-view deconvolution of 5 angles) over a single one angle view. The differences are shown in Fig. 4a-c; in the single angle view the small macromeres (cells A-D) at the vegetal extreme of the embryo are not visible (Fig. 4a). The acquisition of several angles (Fig. 4b and c) reveals the missing cells and makes clear that embryonic 3D reconstructions, which should include all nuclei information, depends on multi-view imaging. A slight improvement of multi-view deconvolution with 12 iterations over average fusion could be achieved (Fig. 4b and c), but appears to be less critical for nuclei staining in early staged M. crozieri embryos. Dual-sided illumination efficiently compensates axial intensity attenuation in semi-transparent specimens Dual-sided illumination for OpenSPIM microscopy can be achieved by building a so-called Tconfiguration, whereby the laser beam gets split into two beams and a second optical path is installed on the optical breadboard. This allows the illumination of specimens from two sides, instead of one, as demonstrated originally by [13], and was also suggested as a potential extension in the original OpenSPIM Page 5 of 16 publication [2]. The benefit of having dual-sided illumination in our OpenSPIM was tested on fixed M. crozieri embryos stained with the nucleic acid marker SytoxGreen. In single-sided illumination images (where one of the two illumination paths has been completely obscured - left or right respectively), a significant loss of signal during acquisition on the side of the missing illumination path becomes obvious due to axial intensity attenuation caused by our opaque and yolky specimens (Fig. 5a and c). The light attenuation is especially apparent when single nuclei from opposed illumination sites (left and right) are directly compared to each other (Fig. 5a and c, insets). In contrast, a more complete picture of the stained nuclei is achieved by using both illumination paths simultaneously (Fig. 5c, insets). This simple test clearly demonstrates the benefit of using dual-sided illumination for our slightly opaque endolecithal polyclad embryos. Two laser lines allow the visualization of two detection channels Our OpenSPIM is equipped with two individual lasers (λ = 488 nm and 561 nm) that allow visualization of two detection channels. The twin laser system was tested on fixed 1 day old M. crozieri larvae stained with the nucleic marker SytoxGreen (488). The 561 laser was used in the same specimens to visualize auto-fluorescence of gland cells (rhabdites). The larvae have gland cell scattered mostly around the apical plate and on the ventral side of the animals, which is shown in Fig. 6 in a single specimen, in which both channels (green and red) have been combined. Here we simply demonstrate the use and precise alignment of the twin laser beams (488 nm and 561 nm). Precise alignment is required during multi Fig. 4 The benefit gained by applying the multi-view deconvolution method (5-angles used) vs. simple maximum projections of an acquired stack (1-angle) on an early staged M. crozieri embryo. a Depth color coding (Fire) of all nuclei after maximum projection of a raw z-stack (1-angle) b Depth color coding (Fire) of all nuclei after a maximum projection of a raw z-stack (1-angle) c Maximum projection of all nuclei after multi-view deconvolution. Additionally all nuclei missing in the single angle maximum projection (from b) have been coloured in red Girstmair et al. BMC Developmental Biology (2016) 16:22 Page 6 of 16 Fig. 5 A single and dual-sided illumination, imaging test showing maximum projections of nuclei stained with the nucleic acid marker SytoxGreen. a Maximum projections of embryonic nuclei using left illumination path of the OpenSPIM microscope, b Both illumination paths and c Right illumination paths respectively. Image stacks were acquired in the following order: left illumination, right illumination, dual-sided illumination (a following c following b). All stacks and related insets have been processed identically color imaging to obtain good quality images from both channels. The initial laser beam alignments in a multiple laser system (in our case VersaLase) is done by the manufacturer and an OpenSPIM user can later only align one wavelength, e.g. 488 nm, while the other wavelength(s) (in our case the 561 nm) is presumed to coincide. It is worth noting that we have transported our OpenSPIM by train and car and that the default alignment of our laser system alignment has proven robust during travelling. OpenSPIM image acquisition with hardware controlled laser triggering is more than twice as fast With the aim of reducing image acquisition time, we incorporated ESio’s TTL controller box (http://www.esimagingsolutions.com/) into our OpenSPIM microscope; Fig. 6 Several angles of a 3D-rendered Müller’s larva showing nuclei in green (captured with the 488 laser detection channel) and gland cells in red (captured with the 561 laser detection channel) Girstmair et al. BMC Developmental Biology (2016) 16:22 this enables hardware-controlled synchronization of the timing of camera exposure and laser triggering. To test our ESio TTL controller, a 100 μm thick single color stack was imaged (1280 × 1080 resolution in 16-bit and an exposure time of 32 ms) with a constant step size of 1.5 μm. In this test, the software-controlled image Page 7 of 16 acquisition (by MicroManager without the TTL controller box) completed the acquisition in 43.5 s. In comparison, when hardware-controlled imaging is used, where lasers are triggered with the TTL controller box from ESio, image acquisition took 17.5 s demonstrating a significant reduction of image acquisition time. Fig. 7 Summary figure of the early embryonic development of the polyclad flatworm M. crozieri (1–128-cell stages) (A-J); SEM pictures (A-J) have been captured for comparison, stills from time-lapse sequences (A-J’), multi-view 3D reconstructions (A-J”); all embryos are shown from animal side. Note that time-lapse images (A-J’) are presented as captured by the OpenSPIM (mirror images) and therefore cleavage direction is opposite to 3D and SEM images. All scale bars are 50 μm Girstmair et al. BMC Developmental Biology (2016) 16:22 Fiji’s bead based registration algorithm and multi-view deconvolution is essential to visualize all nuclei in M. crozieri embryos along the animal-vegetal axis Having the possibility to 3D rotate the specimen and using a faster image acquisition set-up opens up the possibility of carrying out whole-embryo time-lapse videos of the development of an embryo. Fiji’s bead based registration algorithm and multi-view deconvolution plugins [4, 6] make it possible to fuse and deconvolve z-stacks imaged at multiple angles, acquired sequentially at any given time-point. We imaged the early development of Maritigrella covering the spiral cleavage and the formation of the four quadrants (Fig. 7A-J’). To ensure that the development is normal in our live-imaging experiment, we fixed specimens from series of cleavage stages with fixed specimens, for which both 3D reconstructions of immunostained OpenSPIM imaged embryos and SEM imaged embryos were performed (Fig. 7A-J & A-J”). The 3D-reconstructed series of fixed embryos of several stages are also available as 3D models (see Additional file 2). Rapid in vivo time-lapse sequences captured with OpenSPIM show the dynamic early embryonic development of M. crozieri With the OpenSPIM equipped with two illumination paths and capable of rapidly producing, high-quality image stacks, we aimed to visualize the embryonic development in M. crozieri up to the 128-cell stage to further test the potential of OpenSPIM for live-imaging and, ultimately, for lineage tracing. We created a time-lapse sequence of a developing embryo injected at the one cell stage with a nuclear marker (H2B:GFP) and a membrane marker (CAAX:GFP). Our time lapse covers 18 h and shows the stereotypical spiral cleavage and formation of four quartets and further development in M. crozieri. The sequence visualizes the embryo from the animal pole (Fig. 7A-J’ and Additional file 3) and consists of 273 individual time-points. During early cleavage, the live specimens had similar morphology at specific time points when compared with fixed specimens (SEM and 3D reconstructions) at the same developmental stages. Discussion Summary of the capacity of our new OpenSPIM New modifications tested for OpenSPIM When designing microscopes for in vivo imaging with the purpose of tracing cells, one of the goals is to have a high imaging speed in order to have the best time resolution. One of our own modifications included hardware-controlled imaging that appears to be an elegant way of removing unwanted delays during image acquisition. Another relatively new implementation, at least in the context of OpenSPIM, is the use of dual-sided Page 8 of 16 illumination, which we tested on our specimens. We demonstrated that bringing the light-sheet simultaneously from two sides to our opaque and yolky samples results in a significant increase in signal across the sample as shown for the nucleic markers of stained embryos (Fig. 5). Limitations of our self-built OpenSPIM It is worth mentioning that simultaneous dual-sided illumination can lead to the light-sheet widening and a reduction in image quality due to additional light scattering effects and shadowing [13]. In our sample, these issues do not outweigh the benefits gained by dual-sided illumination in comparison to the otherwise much more severe attenuation effect observed, but it is assumed to have an impact on the final image quality. Another limitation of conventional light-sheet microscopy concerns the thickness of the light-sheet. Thinner light-sheets are governed by the illumination objective and the thickness depends in particular on their numerical aperture. Ideally the thickness of the light-sheet is uniform across the field of view. In reality, it widens on each side of view and its narrowest point resides in the middle. Fortunately, that is where the sample is normally located. When a static light-sheet is created by passing a pencil beam through a cylindrical lens, the ratio of the light-sheet thickness between the center and the side of the field of view depends on the numerical aperture of the illumination objective and the size of the field of view, i.e. the magnification of the detection objective. OpenSPIM as described presents a good compromise, however the sample should be positioned as centrally as possible for optimal sectioning. To address these problems in light-sheet microscopy, and of particular importance for specimens larger than our embryos, more advanced light-sheet microscopes illuminate the sample from left and right sides sequentially (rather than simultaneously as presented here (see Fig. 5) and also take advantage of pivoting (scanning) the light-sheet as described for the mSPIM [13]. This significantly reduces scattering and attenuation across the field of view. Alternatively, a light-sheet can be generated by scanning a Gaussian beam up and down across the field of view [14]. Thus, the light-sheet that is created can be further modified in various ways [15, 16]. However, such a light-sheet formation paradigm goes beyond the original OpenSPIM design. Nevertheless it can be implemented on the OpenSPIM platform and it is expected that the community of users forming around OpenSPIM will do so. Points for consideration before purchasing and building an OpenSPIM There is little doubt that a self-built light-sheet microscope is significantly more affordable than existing Girstmair et al. BMC Developmental Biology (2016) 16:22 commercially available alternatives. A laboratory considering whether to embark on building one ought to consider two factors. First is the question of whether the finished microscope will be an adequate alternative in terms of image quality and ease of use for the specific task. Second, it is essential when considering an OpenSPIM to factor in the hidden costs involved, most obviously the costs implied by the time spent building the microscope, learning to use it and learning to use the open source software required to run the microscope and to process the data acquired. For our purposes the quality and speed of acquisition that we were able to achieve with our homebuilt OpenSPIM device provides valuable, high quality data that suit our requirements. However, despite the fact that the assembly of an OpenSPIM is indeed quite straightforward, this step remains only one of the many challenges to overcome. Here we summarize various steps we feel are worth considering before building an OpenSPIM in order to avoid assembling an expensive toy that will be forgotten shortly after (Fig. 8). Before you begin Before beginning we would recommend prospective OpenSPIM users to image your own specimens on an established OpenSPIM system. This will provide valuable information on whether the system will be suitable for your purposes as well as show what is required in terms of hardware for capturing highquality images of your particular specimens. This is also an opportunity to gain skills such as correctly aligning the light-sheets, getting familiar with the acquisition software, finding the optimal mounting strategy for the specimens and will inform decisions for the OpenSPIM design selected, as discussed in the next section. There are many OpenSPIM systems around the world; the current estimate is 70. The system from Tomancak lab also regularly travels to practical courses and was extensively used during the EMBO course on Light-sheet Microscopy in Dresden (August 2014 http://openspim.org/EMBO_ practical_course_Light_sheet_microscopy and upcoming August 2016). Page 9 of 16 Designing your OpenSPIM The basic design of an OpenSPIM can be taken from the open access platform (http://openspim.org). However, modifications, which might meet more specific needs of individual users, require well thought-through decisions especially considering that most divergences from the basic plan will involve higher costs and, very likely, additional trouble shooting. We have discussed the most significant amendments we have made in building our own OpenSPIM. Dual sided illumination allows us to image our relatively opaque, yolky embryos optimally and we have shown the benefits of this. Twin lasers allow us to observe more than one labelled molecule per sample. As the most expensive component, the laser system is of particular importance, especially when it comes to multi-channel acquisition. Our OpenSPIM can be easily upgraded up to as many as 4 different laser wavelengths whose beams are aligned within the laser system itself. This alignment of two lasers has proven robust even when travelling, meaning the microscope is fairly portable. As our embryos are extracted from animals living in tropical waters they can be left at room temperature (23° C) during development. Therefore our OpenSPIM chamber temperature is currently not temperature regulated, but depending on the experiment or the specimens used for imaging a more sophisticated control of the chamber temperature might often be advantageous or even a requirement. While we have no experience of controlling variables in the acquisition chamber, we think that temperature control could be achieved easily by simply placing the chamber on top of a heating/cooling plate. Additional regulations such as pH or CO2 controls, although feasible, would require more elaborate chamber modifications to avoid, for example, bubbling or flow disturbance of the chamber water created by the connected gas supply and reliable measuring systems. Time taken for purchasing While this might sound trivial, we found that purchasing elements occupied a significant time. There were three reasons for this, first that there is a multitude of suppliers to be negotiated with. Second, as some of the parts are expensive we (like many institutions) were required Fig. 8 Flow chart illustrating steps necessary for establishing a home-built OpenSPIM Girstmair et al. BMC Developmental Biology (2016) 16:22 to obtain multiple quotes for each item. Third, the choices made regarding some parts had knock-on effects regarding the choice or specification of other parts. Finally it should not be forgotten that some of the parts of the OpenSPIM are bespoke and require a workshop or manufacturer for their production. Assembly The assembly of all parts can be considered fairly easy, also thanks to the information provided from the http:// openspim.org website. Usually this should be the least time consuming (and the most fun) task. Software and hardware integration The MicroManager software and information provided on the OpenSPIM website makes correct hardware configuration relatively easy. However - at least in our experience - establishing the correct links between hardware components and the acquisition computer causes time consuming problems. Additional time for hardware testing and configuration should be allowed. As an example, we experienced a major issue installing a simple FTDI chip driver, which is necessary for the ESio’s TTL controller box to communicate with the acquisition computer. Solving this problem required additional testing of the hardware and interaction with the original suppliers. Moreover, we strongly recommend interacting with the growing OpenSPIM online community via the mailing list, since many users are experiencing the same problems and it is the power of this community that will help you overcome them. Besides, hardware software integration is not something that a typical biologist can master with ease. Involving computer scientists or engineers on the undergraduate level, which should be relatively easy at any large University, is likely to smooth many integration problems. It will also give the students valuable experience with open access hardware and electronics and connect them with the active online communities in these areas. A fast way to correctly align the OpenSPIM Learning how to correctly align the light-sheet is a skill that might require some help, but it is not particularly difficult to learn. Within the materials and methods we describe what we learned and how we currently align our two excitation light-sheets by simply adjusting the 25 mm and 50 mm telescope lenses and the two adjuster knobs (Horizontal & Vertical) of the Gimbal mounts of each corner mirror. It is a relatively fast approach (and certainly not the only one), but in our experience the results achieved in terms of image quality are more than satisfying. Page 10 of 16 Time needed to complete building the OpenSPIM Altogether it took us about 7 months from ordering the OpenSPIM parts to acquiring a first image. This time span is surely highly variable and some delays we experienced and mentioned above (see time taken for purchasing) can probably be improved or avoided at all. Image processing Imaging and processing and the challenge of multi-view 4D microscopy of acquired data can be straightforward or become a major issue depending on the operator’s ambitions. Acquisition of z-stacks of fixed specimens and subsequent processing with Fiji can be easily learned and more sophisticated processing such as multi-view deconvolution can be learned using online Fiji tutorials on how to use the necessary plugins. Considerably more challenging, in our experience, is long-term multi-view 4D microscopy of live embryos. This live-imaging setup (keeping the embryo alive and developing normally during acquisition for example) is clearly important. It is also essential to consider the challenge of post-processing the huge amount of data that are generated. Home-built OpenSPIMs are in principle capable of creating elaborate multi-view 4D microscopy videos on difficult specimens (e.g. opaque embryos with scattered emission-light). This was successfully demonstrated on Drosophila embryos, where data from six angles per time point have been acquired and the OpenSPIM data generated were subsequently successfully reconstructed using Fiji’s bead based registrations algorithm and fused via multi-view deconvolution. However, multi-view 4D microscopy requires an efficient work flow saving the produced data onto hard drives. The second major requirement is a precise 4D motor system to keep the positional information of the specimen over time as exact as possible in concert with acquisition software that allows the correction of minor drifting of the specimen. Finally major computational resources are needed for processing the data generated, especially if multi-view deconvolution of hundreds of time-points is intended. In our opinion multi-view 4D microscopy is one of the most demanding and challenging experiments one can undertake with a home-built OpenSPIM and will thus be discussed further in the following section. Inefficient data saving can prolong time-point intervals during imaging Multi-view 4D microscopy requires software that reliably saves large amounts of data on hard-drives without running into the problem of a data bottleneck. In singleview time-lapse videos, we observed that the creation of closely spaced time-points (intervals from about 90 s/ time-point) with the MicroManager SPIMacquisition