Name: Upright Imaging of Drosophila Embryos (Video) | JoVE Journal
Uploaded: 2010-09-13T17:00:00.000+00:00
Duration: 7 min 34 s
Description: Learn upright mounting of Drosophila embryos using glycerin jelly for confocal cross-sectional imaging. This method enables precise measurement of embryo size and quantification of protein and RNA expression levels.

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5 minutes

The authors are especially indebted to Deborah Harris and Danielle MacKay. Support for this work was provided by the Department of Biology and the College of Arts and Sciences of CWRU, and by HHMI grant number 52005866 for support of undergraduate education in the biological sciences.

1. Imaging Methodology
<ol>
	<li>Melt glycerin jelly in a 55° C degree water bath for 20-30 minutes until it becomes a homogenous liquid. Swirl, but do not shake the bottle to avoid the formation of bubbles.</li>
	<li>While the jelly melts, apply a thin layer of glycerol using a Kimwipe on a clean glass slide (optional).</li>
	<li>Pipette 1 to 1.5 mL of melted glycerin jelly using a cut P1000 pipette tip on the surface of the slide to create a thin first layer. Pipette carefully to avoid bubbles.</li>
	<li>Let the jelly solidify for 5 min on a horizontal surface, then place the slide at -20°C for 5 min and proceed to embryo alignment. Alternatively, cover the slide with Saran Wrap and place it at 4°C for up to 5 days.</li>
	<li>Pipette a small drop of stained embryos in 70% glycerol/PBS or other antifade glycerol-based mounting media (e.g. Slowfade or Vectashield) on the side of the jelly bedding.</li>
	<li>Begin pre-aligning the embryos by individually transferring them from the drop to the jelly surface. Transfer them using a slanted hypodermic needle, using the slanted side as a spoon.</li>
	<li>Roughly place the embryos along 2 to 3 columns. If an embryo gets stuck in the needle, stir it in the drop containing the other embryos to release it. Do not waste time carefully aligning them at this point. Leave some space between columns.</li>
	<li>Align the embryos by sliding them horizontally on the jelly medium with the aid of the hypodermic needle. While sliding, orient the heads and tails in the same direction. Make them parallel to each other along the column. Remove excess of PBS glycerol left as a trail with a piece of twisted Kimiwipe. Note: Excess glycerol will cause embryos to be loosely encased within gel, resulting in separation of the two jelly layers, making it difficult to cut and mount. Conversely, removing too much glycerol will dry and flatten embryos.</li>
	<li>Use a cut yellow tip to spread a thin layer of jelly over the embryos (50-150 μL, depending on how many were aligned).</li>
	<li>Place slide in -20°C freezer for 10-20 min or store it at 4°C overnight. Make sure the jelly is completely rigid before cutting. Otherwise, the block of embryos will be very difficult to excise. Best results for cutting are obtained on the following day.</li>
	<li>Under a dissecting scope, use a razor blade to cut completely through the jelly on both sides of the aligned embryos. Hold the razor blade at a 90° angle to make straight cuts. The cuts should be made very close to the anterior and posterior poles of the embryos to avoid excess jelly that may obstruct imaging.</li>
	<li>Add 100-200 μL of cold PBT (4°C, PBS+0.1% Tween 20) next to the cuts and carefully scrape off the jelly between embryos using a spatula. Note: The detergent in PBT helps the removal of the jelly from the slide without damaging the embryos.</li>
	<li>Using a needle, transfer the block of jelly with embedded embryos to a clean slide for further cutting. Cut smaller blocks with 5-7 embryos. Use PBT to easily move around the jelly block. Alternatively, place the block on a dry glass surface to let it attach to the glass, which creates more stability for precisely trimming the jelly, if necessary.</li>
	<li>Flip upright the embryos embedded in the jelly with the aid of a razor blade and a needle. For visualization under microscope, proceed with either one of the two steps below, depending on the type of microscope stand being used.</li>
	<li>Inverted microscope: place finished embryo blocks onto a long coverslip, with embryos in an upright position. Ensure that the side of the embryo with the least amount of jelly will be in closer contact with objective.</li>
	<li>Upright microscope: make two stacks with four #1 coverslips glued with superglue to be used as "supports" and place the embryo block in glycerol in between the "supports". Bridge the stacks using a long coverslip.</li>
	<li>For confocal analysis, check the working distance of the objectives to be used to find out whether you can image all the way through the embryo or only partially. For example, D. melanogaster embryos have ~ 0.55 mm in length and can be entirely imaged using a Zeiss 20x Plan-Apo or a 40x LD Neo-Fluor objective. The following website has information on available Zeiss objectives and gives you the option to search objectives according to working distances: https://www.micro-shop.zeiss.com/us/us_en/objektive.php</li>
</ol>
2. Representative Results
Because the embryos are left intact, this method can be used to take precise measurements of embryo size and distances between gene expression patterns. In Figure 1, we show a blastoderm stage embryos stained with the nuclear stain DAPI (blue), anti-Dorsal protein (red), sog mRNA (yellow) and sna mRNA (green). Morphological processes, such as invagination of the mesoderm (Figure 2A, B) and migration of mesodermal cells (Figure 3 and 4) can also be visualized using this method. Different Z-positions along the DV axis can be obtained by changing the focal plane, as shown in Figures 2-4.
<img src="/files/ftp_upload/2175/2175fig1.jpg" alt="Drosophila embryo fluorescence microscopy; mRNA, protein, nuclei distribution; gene expression analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Upright optical slice of an intact embryo in blastoderm stage. Double staining for mRNA and protein. Target mRNAs for in situ probes (sog and sna) and protein stained by primary antibody (anti-Dorsal) are indicated in the figure. DAPI was used to label the nuclei. Note that the expression of sog and sna are located apically in the cytoplasm, while the expression of Dorsal is nuclear. Strong signals from sog nascent transcripts that are located in nuclei can also be seen at this magnification.
<img src="/files/ftp_upload/2175/2175fig2.jpg" alt="Gene expression fluorescence microscopy; diagram; sog, sna, ind genes; DAPI staining; focal planes." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Upright imaging of an intact gastrulating embryo. mRNA probes used and their respective colors are indicated in the figure. A) Note that in this stage, three genes stained with same fluor (sna, sog and dpp, in green), are expressed in spatially separated domains. The invaginating mesoderm expresses sna, the neuroblast layer expresses ind and the ectoderm expresses dpp, while sog expression is still present in ventral regions of the nervous system. B) In a deeper confocal plane of the same embryo, we note the base of the invaginating mesoderm strongly expresses sna, but its apical region expresses lower levels.
<img src="/files/ftp_upload/2175/2175fig3.jpg" alt="Gastrulation process diagram; focal plane images A and B; showing neuroectoderm, mesoderm layers." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Upright imaging of an intact embryo with germ band extended. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). Nuclei were stained with DAPI (blue). A) Focal plane near embryo head. B) Another focal plane in the trunk region of same embryo. Note mass of mesodermal cells before lateral migration (compare to Figure 4) lying close to the ventral midline on both sides of a germ-band extended embryo. Delaminating neuroblasts are labeled in green (ind and sna).
<img src="/files/ftp_upload/2175/2175fig4.jpg" alt="Confocal microscopy images, trunk and mid-posterior regions, fluorescence marking cellular structures." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Upright imaging of an intact embryo during germ band extension. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). A) Note the delaminating neuroblasts from the epithelium (arrow), stained both for ind and sna at this stage (green). Note also the restricted expression of sog to the ventral midline (yellow). Expression of dpp at this stage can be seen at lateral sides of the embryo (asterisk). B) Optical section in the same embryo shown in A near the end of the embryo length, which corresponds to mid-posterior region. The ventral midline cells are stained for sog.

<ol><li>Ay, A., Fakhouri, W. D., Chiu, C., Arnosti, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image+processing+and+analysis+for+quantifying+gene+expression+from+early+Drosophila+embryos%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">Image processing and analysis for quantifying gene expression from early Drosophila embryos.</a> Tissue Engineering. 14, 1517-1526 (2008).</li>
<li>Grβhans, J., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+link+between+morphogenesis+and+cell+division+during+formation+of+the+ventral+furrow+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila.</a> Cell. 101, 523-531 (2000).</li>
<li>Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+detection+of+RNA+expression+in+Drosophila+embryos%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li>
<li>Witzberger, M. M., Fitzpatrick, J. A. J., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((End-on+imaging%3A+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Developmental Dynamics. 237, 3252-3259 (2008).</li>
<li>Zander, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+mounting+delicate+bryophytes+in+glycerol%5BTitle%5D)+AND+%22The+Bryologist%22%5BJournal%5D)">On mounting delicate bryophytes in glycerol.</a> The Bryologist. 100, 380-382 (1997).</li>
<li>Liberman, L. M., Reeves, G. T., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+imaging+of+the+Dorsal+nuclear+gradient+reveals+limitations+to+threshold-dependent+patterning+in+Drosophila%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila.</a> Proc Natl Acad Sci U S A. 106 (52), 22317-2222 (2009).</li>
</ol>

No conflicts of interest declared.

Taking cross section images of Drosophila embryos can be challenging, since it requires either a careful hand dissection of slices 2 or the use of embedded media for microtome processing, which is usually detrimental for fluorescent stainings. Alternatively, Z-stacks made in a Confocal microscope can be used to make 3D reconstruction of cross-section images. However, it is time consuming to make several thin Confocal slices for an accurate 3D reconstruction and photobleaching becomes problematical. Another concern when imaging embryos that are mounted longitudinally is the light scattering that occurs when reaching deeper sections of the embryo, which also complicates taking precise measurements of fluorescent signals for the purpose of quantification of protein or mRNA expression levels1. Even with a two-photon confocal microscope, light scattering is still a concern due to the opacity of yolk material in the middle of the embryo, which makes selection of mounting media for optimal transparency of sample to be limited.
To circumvent these problems, a new technique called "End-on" imaging of positioning embryos vertically was recently developed to image both live and fixed samples 4. In this paper, we developed a new protocol for vertically mounted embryos that allows a simple preparation and visualization of fixed embryos or tissues of any size and shape that are stained with multiple in situ probes 3. Our method consists of using a mounting media with gelatinous consistency that is used to encase aligned embryos within it. Cut blocks of this mounting media containing embedded embryos can be positioned upright before imaging in any type of Confocal microscope. 
By embedding the embryos into the jelly and positioning them vertically, we are able to take horizontal cross-sections using Confocal microscopy, in which cells along the dorsal-ventral axis of the embryo are presented simultaneously. This is a significant improvement for quantification purposes since problems of light scattering and photobleaching of fluorescent dyes that occurs in conventional Z-stack reconstruction of longitudinally mounted embryos are now eliminated. Thus, quantification of gene expression or protein levels along the dorsal-ventral axis is accurate, since cells located at opposite dorsal-ventral positions are imaged at the same time and at the same confocal Z-position. In addition, the described method allows us to obtain a complete 2-D image across the dorsal-ventral axis from a 3-D embryo without using complex computational unrolling methods to visualize cells at different positions along the DV axis 6.
Some of the critical steps include removing excess glycerol after aligning embryos, to avoid split between the two layers of jelly. However, if the sample is too dehydrated, the resulting image will be misshapen and have incorrect morphology. In addition, if the jelly medium around the embryo is cut at an angle rather than straight, the resulting image may appear less round and more ovular in shape, due to an incorrect positioning of the embryos at an angle other than 90 degrees. Finally, if the jelly is not cut closely enough to the embryo on the anterior/posterior ends, it is not possible to obtain a proper image because the objective's working distance would be mainly used to focus into the jelly and not the embryo.
Another important step that may be necessary when imaging late gastrulating embryo is to carefully sort the stages of interest under the scope before aligning them. Usually, embryos are staged according to morphological characteristics that can be most easily recognized when in a longitudinal position.
Among alternative modifications that can be made with this method is to include an anti-fade reagent (e.g. p-Phenylenediamine or PPD) into the second layer of jelly to help protect against photobleaching of fluorescent dyes.

<table><tbody><tr><td>Glycerin Jelly mounting media</td><td>Electron Microscopy Sciences</td><td>17998-10</td><td>Contains low concentration of phenol as preservative and should be used in hood or in well ventilated area. Recipe for making your own media is described in Zander, 1997.</td></tr><tr><td>Zeiss LSM 700 Confocal</td><td>Carl Zeiss, Inc.</td><td></td><td>The following objectives were used: Plan-Apo 20x 0.8 M27 (WD 0.55mm); EC Plan-Neofluar 40x 1.3 oil (WD 0.21mm); LD Plan-Neofluar 0.6 Korr 40X (WD 2.9mm).</td></tr><tr><td>Phosphate buffered saline with 0.1% Tween 20 (PBT)</td><td></td><td></td><td></td></tr><tr><td>Glass microscope slides</td><td>Fisher Scientific</td><td>12-544-7</td><td></td></tr><tr><td>Glass cover slips</td><td>Electron Microscopy Sciences</td><td>72204-02</td><td>Size 1 &amp;#189; (specific for the objectives used in this work).</td></tr><tr><td>Glass cover slips (for making supports)</td><td>Fisher Scientific</td><td>12-544-10</td><td>Use four coverslips glued with superglue. </td></tr><tr><td>Dissection microscope</td><td>M5 Wild Heerbrugg Wild Makroskop</td><td>M420</td><td></td></tr><tr><td>Razor blade</td><td></td><td></td><td>Steel back single edge industrial blades</td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>21-401-10</td><td></td></tr><tr><td>Hypodermic needles</td><td>Fisher Scientific</td><td>1482610</td><td>26 Gauge</td></tr><tr><td>Pipettes</td><td>Labnet International</td><td></td><td></td></tr></tbody></table>

upright imaging of drosophila embryos

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

The overall goal of the following experiment is to obtain coronal viewing images of intact oph embryos using confocal microscopy that can be used to precisely measure embryo size and quantify protein and RNA expression levels. This is achieved by using a mounting media of gelatinous consistency to encase embryos within it and position them upright. As a first step.The embryos are aligned over a thin layer of glycerin jelly and then covered with a second layer of this media. Next, the mounting media containing the embryos is cut into blocks, which can be flipped vertically to hold. The embryos in position for imaging results are obtained that show cross-section images visualized with any sort of confocal microscope.Hello, my name is Milu. I'm a graduate student at the Meso Lab in the biology department at Case Western Reserve University. Today we are going to demonstrate to a mounting protocol to image PHI embryos in an abroad position using a confocal microscope.We developed this method during our developmental biology course lab here at Case in collaboration with Dr.The main advantage of this technique over existing methods like the Endon imaging technique developed by the Mindin Laboratory, is that it allows for simple preparation and visualization of fixed embryos or tissues of any size and shape. We first had the idea for this method when we needed to visualize cross sections of very small embryos of other gesso species, which would be very difficult to hand slice. This method can help answer questions in the developmental biology field by allowing precise quantifications of gene expression and protein levels along the dorsal ventral axis.Typically, embryos are mounted longitudinally. While it's possible to get Zack reconstructions of cross-sections, there's often a problem of photobleaching of the fluorescent eyes light scattering, and it is very time consuming. Our technique minimizes these problems because cross-sectional images of embryos are visualized along a single confocal point.Generally, individuals new to this method may experience some difficulties while cutting the glycerin jolly blocks near to tip of the embryos. Visual demonstration of this method is critical because it requires careful handling. We have a demonstration today made by our students, Catherine Chen and catty To prepare drosophila embryos for upright imaging.First melt glycerin jelly in a 55 degree Celsius water bath for 20 to 30 minutes until it becomes a homogenous liquid swirl. But do not shake the bottle in order to avoid the formation of bubbles. Using a cut P 1000 pipette tip carefully pipette one milliliter of melted glycerin jelly onto the slide.To create a thin even first layer free of air bubbles, let the jelly solidify for five minutes on a horizontal surface. Then place the slide in a minus 20 degree Celsius freezer for five minutes. Alternatively, cover the slide with plastic wrap and place it at four degrees Celsius for up to five days.Once the first layer of jelly is solidified, bring the slide to the scope and proceed to embryo alignment. Pipet a small amount of stained embryos and 70%PBS glycerol on the side of the first layer of jelly. Under the scope, align the embryo side by side along three to four columns per slide.Use a hypodermic needle to scoop them using the slanted side of the needle. As a spoon. Carefully orient the heads of all embryos in the same direction by sliding them horizontally on the jelly medium with the aid of the needle.The final configuration of the aligned embryos will be similar to that described for DNA injection. Using a twisted piece of Kim wipe, remove excess PBS glycerol from embryos. Carefully pipette between 50 to 300 microliters of melted glycerin jelly over the embryos, depending on the number of embryos to make a second layer of jelly.Finally, cool down the slide at minus 20 degrees Celsius for five to 10 minutes, or store it at four degrees Celsius overnight. To remove the glycerol embedded embryos from the slide for imaging under a dissecting scope, use a razor blade to cut through the jelly in close proximity to the anterior and posterior sides of the aligned embryos. Hold the razor blade at a 90 degree angle.To make straight cuts, add 100 to 200 microliters of cold PBT next to the cuts. To carefully scrape off the jelly next to the embryos. Using a spatula, PBT helps release the block of embryos.Transfer the block to a dry microscope slide for further cutting. First cut smaller blocks with five to seven embryos within it. Second trim excess of glycerin jelly that may interfere with imaging.Finally, flip the embedded embryos to an upright position with the aid of a razor blade and a needle. To visualize drosophila embryos using an inverted confocal microscope, place the jelly block containing embryos onto a cover slip. Ensure that the site of the embryo with the least amount of jelly is in closer contact with the objective.If using an upright microscope to image, make four stacks of supra glue. Number one, cover slips to be used as supports and place the embryo block in glycerol in between the supports. Bridge the stacks using a longer cover slip for confocal analysis.Place the specimen on the microscope stage. Check the working distance of the objectives to be used to find out whether you can image all the way through the embryo or only partially set up the confocal configuration in the computer to start imaging as shown here. If the protocol is done correctly, cross-section images of embryos with intact morphology can be observed because the embryos are left intact.This method can be used to take precise measurements of embryo sizes and gene expression levels across the dorsal ventral axis. Once mastered, this technique can be done in 30 to 45 minutes. It is important to remove excess glycerol before pouring on the second layer of glycerin jelly over the embryos.Otherwise, the two layers may split and make the cutting step More difficult. Also, you need to check the working distance of the objectives you're using to make sure that you can image the entire embryo or only part of it because the working distance creates a limitation of how much you can image. Make sure to cut the jelly closely to the side of the embryo that you will be positioned towards the objective.Otherwise, you'll be imaging only jelly instead of the embryos. After watching this video, we hope you'll have a good understanding how to take cross-sectional images of Joseph Embryos. Thank you for watching us and good luck with your experiments.

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Learn upright mounting of Drosophila embryos using glycerin jelly for confocal cross-sectional imaging. This method enables precise measurement of embryo size and quantification of protein and RNA expression levels.

Upright Imaging of Drosophila Embryos

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