Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila larva body wall is composed of 30 identifiable muscle fibers arranged in a regular pattern and looks like a thin slice after dissection and fixation²¹(Figure 1A). The sample remains flat during the dehydration process due to the presence of the metal meshes (Figure 1B, C). The larval body muscle is buried in a thin plate made of epoxy resin (Figure 1D–G), which is easily and accurately positioned and glued to the resin-filled capsule (Figure 1H, I).

Figure 1: Schematics of TEM sample preparation of Drosophila larva body wall. (A) Samples made after dissecting, fixing, and rinsing of Drosophila larva. The sample is placed in (B, B') a flat-bottomed test tube and (C) is dehydrated and infiltrated while being sandwiched between two metal meshes. The sample is transferred into (D, E) a pre-made polyethylene embedding plate, (F) in which it is embedded and polymerized. (G) After initial engraving of the sample, thereby removing excess resin, (H, I) the engraved sample is glued to a resin-filled capsule. Scale bar, A: 4 mm; B': 2.5 cm; D-G: 2 mm; H, I: 3 mm. This figure has been modified from Guangming, et al.¹⁶. Please click here to view a larger version of this figure.

As the neuromuscular junction is located in the narrow region between the 6th/7th muscles²² (Figure 2A), we opted for a longitudinal incision along the muscle. Excess resin and muscles near the target regions were removed²³. After fixation with OsO4, the samples exhibited a dark brown coloration. The samples with epoxy resin required a sequential polymerization process at 37 °C (24 h), 42 °C (24 h), and 60 °C (24 h). The samples with Lowicryl K4M resin were polymerized in UV light with 365 nm wavelength for 3 days at − 35 °C, followed by polymerization for 3 days at 25 °C. The Lowicryl K4M resin presented a white and transparent appearance (Figure 2B), while the epoxy resin appeared yellow (Figure 2C). When examining the body muscles, it was found that the use of Lowicryl K4M resin provided sharper and more easily observable results compared to the epoxy resin. However, it was easier to perform the procedures with epoxy resin at room temperature. Epoxy resin also permitted precise positioning. (Figure 2B–K).

Figure 2: The trimming process of NMJ boutons between 6^th/7^th muscles in Drosophila larva. (A) shows 6^th and 7^th intermuscular NMJ immunostaining pattern, where type Ib NMJ buttons (large arrows) and type Is NMJ buttons (small arrows) can be observed. Hrp antibody and Dlg antibody labeled the presynaptic and postsynaptic structures of type I NMJ boutons. Drosophila larvae samples were embedded in (B) Lowicryl K4M resin and (C) Epon 812 resin, respectively, and the 6^th and 7^th muscles were observed under light microscopy. The blue arrows show the connections between body sections. (D) Serial ultrathin sections were performed in the specific area between the 6^th and 7^th muscles: the red line is the beginning line, and the green line is the end line. (E) The A₂, A₃ and A₄ body segments of Drosophila larva can be clearly observed under the light microscope after the embedding block was roughly trimmed with a glass knife. (F) Here the A₃ segment was retained and trimmed. (G-J) Remove excess resin and (K) unnecessary cuticle near the A₃ segment. Scale bar, B, C: 80 µm; E-K: 300 µm. This figure has been modified from Guangming, et al.¹⁶. Please click here to view a larger version of this figure.

It is difficult to see the Drosophila larval NMJ samples processed by traditional sample preparation procedures for electron microscopy^14,15. However, based on the distribution pattern of Drosophila larval NMJ under confocal (Fig. 3A–A'), it was hypothesized that boutons between the 6^th/7^th muscles could be observed more easily by the new method. The neuromuscular junctions can be observed in the form of beads under electron microscopy (Figure 3B–B', D', E-E'). In addition, this new method provided us with more information about the synaptic structure under electron microscopy.

Figure 3: Electron microscopic images of Drosophila larva NMJ. (A, A') After removing the unnecessary resin and cuticle near the 6^th muscle, the thickness of the remaining sample was about 100 µm. (B, D) Successive sections were performed starting from the 6^th muscle, and as successive sections progressed, the resin component became more abundant (B') while synapses began to gradually appear. Two consecutive sections were observed under electron microscopy, and type Ib synapses were observed in the superficial layer of the 6^th muscle with synaptic vesicles, postsynaptic membranes (thin white arrows), presynaptic membranes (thick white arrows) with attached T-bars (white wedge) and dense SSRs (D, D'', E, E'''). The thick black arrows show trimmed muscle and cuticle, and the thin black arrows show the trimmed resin margins. Scale bar, A, A', B, C, D, E: 10 µm; B', D', E'': 200 nm; E': 500 nm; D'', E''': 50 nm. This figure has been modified from Guangming, et al.¹⁶. Please click here to view a larger version of this figure.