Name: using light sheet fluorescence microscopy to image zebrafish eye development
Uploaded: 2016-04-10T15:00:00
Duration: 13 min 1 s
Description: Watch this Scientific Journal Video about Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development at JoVE.com

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 border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Lightsheet Z.1 microscope</td>
			<td style="width:184px;">Carl Zeiss Microscopy</td>
			<td style="width:119px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting point agarose</td>
			<td>Roth</td>
			<td>6351.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting point agarose</td>
			<td>Sigma</td>
			<td>A4018 or A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate (MESAB/MS-222/Tricaine)</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td>Sigma</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">500 nm red fluorescent beads F-Y 050</td>
			<td>Millipore (Estapor)</td>
			<td>80380495</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 &#956;l (1mm inner diameter, marked black) capilllaries</td>
			<td>Brand</td>
			<td>701904</td>
			<td>sold as spare part for transferpettor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Teflon tip plungers for 20 &#956;l capillaries</td>
			<td>Brand</td>
			<td>701932</td>
			<td>sold as spare part for transferpettor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips diameter 18 mm, selected thickness 0.17 mm</td>
			<td>Thermo scientific (Menzel-Glaser)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O-rings for chamber windows 17&#215;1.5 mm</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezers, style 55</td>
			<td>Dumont</td>
			<td>0209-55-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Luer-Lock syringes</td>
			<td>Becton Dickinson</td>
			<td>300865</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 cm extension cable for infusion compatible with Luer-Lock syringes</td>
			<td>Becton Dickinson</td>
			<td>397400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Links</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 1 Multiview reconstruction application</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/SPIM_Registration http://fiji.sc/Multiview-Reconstruction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 2 BigDataViewer</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 3 BigDataViewer</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc/BigDataViewer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 4 Multiview reconstruction application-issues</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/SPIM_Registration/issues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 5 BigDataViewer-issues</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/bigdataviewer_fiji/issues&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 6 BigDataServer</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc/BigDataServer.</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/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/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/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/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/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/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 ...

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