Upright Imaging of Drosophila Embryos

1. Imaging Methodology

1. 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.
2. While the jelly melts, apply a thin layer of glycerol using a Kimwipe on a clean glass slide (optional).
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. Use a cut yellow tip to spread a thin layer of jelly over the embryos (50-150 μL, depending on how many were aligned).
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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

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.

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.

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.

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).

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.