The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology and responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, longer-term live imaging has been technically challenging because of a lack of efficient methods for immobilizing larvae for extended periods. We describe here a simple procedure for anesthetization and uninterrupted long-term in vivo imaging of the epidermis and other larval organs, including gut, imaginal discs, neurons, fat body, tracheae, muscles and hemocytes, for up to 8 h. We also include a procedure for probing cell properties by laser ablation. We provide a survey of the effects of different anesthetics, demonstrating that short exposure to diethyl ether is the most effective for long-term immobilization of larvae. This protocol does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and subcellular processes in larvae.
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We are grateful to M. Kakanj for her photographic and graphical support. We thank S. Roth, N. Riddiford, A. Schauss, F. Papagiannouli, V. Böhm and C. Lesch for critical reading of manuscript, comments and helpful discussions. We thank A. Schauss, P. Zentis and C. Jüngst from the CECAD imaging facility in Cologne (University of Cologne, Cluster of Excellence in Ageing Research) for support and the Bloomington, VDRC and DGGR stock centers for fly strains. This work was supported by grants from the European Regional Development Fund and the German state North Rhine-Westphalia (NRW im Ziel 2) to S.A.E. and L.P., a CMMC grant to M.L. and S.A.E., a CECAD grand by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy, grant EXC-2030/1-390661388 to M.L. and an EMBL research fellowship to P.K.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Greg Macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Kakanj, P. et al. Nat. Commun. 7, 12972 (2016): https://doi.org/10.1038/ncomms12972
Beati, H. et al. J. Cell Biol. 217, 1079–1095 (2018): https://doi.org/10.1083/jcb.201610098
Live imaging of mitochondria (green) dynamics during epidermal wound healing. Single-cell wound in the dorsal epidermis of Drosophila L3 larva expressing mito-GFP (green) and DsRed2-Nuc (magenta) to mark mitochondria and nuclei in the epidermis: A58>mito-GFP; DsRed2-Nuc (w1118; UAS-mito-HA-GFP/+; A58-Gal4, DsRed2-Nuc/+). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 7a-b.
Live imaging of ER vesicle (green) trafficking between ER and Golgi in the epidermis of Drosophila L3 larva expressing e22c>; RFP-KDEL (w1118; e22c-Gal4/+; UASp-RFP.KDEL/+). KDEL is an ER marker, encodes a sequence to prevent secretion of the protein form ER or retrieval of ER proteins from the Golgi. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7c-d.
Live imaging of fat body (green) in the L3 larvae expressing c7>mCD8-GFP (w1118; c7-Gal4/+; UAS-mCD8-GFP/+). The mCD8-GFP marks the cell membranes. Each frame is a single plane. Scale bars: first movie 80 µm and second movie 20 μm. Corresponds to Fig. 7e-f.
Live imaging of trachea (green) in the early L3 larvae in two individuals both expressing btl>GFP (w1118; btl-Gal4, UAS-GFP; +). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bar: (a,b) 17 μm. Corresponds to Fig. 7g-h.
First movie shows a live imaging through the z-stacks of dorsal compartment of wing imaginal disk (grey) in late L3 larva expressing hh>mCD8-GFP (w1118; +; hh-Gal4, UAS-mCD8-GFP/+). The mCD8-GFP marks the cell membranes. Each frame is a single plane of z-stacks (54 stacks with 1µm space). Scale bar: 50 μm. Second movie shows a short live imaging of entire imaginal disk (green) in early L3 larvae expressing esg>GFP (w1118; esg-Gal4, UAS-GFP/+; +). Each frame is a single plane. Scale bar: 100 μm. Corresponds to Fig. 7i-k.
Live imaging of peripheral neuron (black) in the L3 larva expressing mCD8-GFP (black) to mark cell membranes of the neurons: elav>mCD8-GFP (w1118, elave-Gal4/UAS-mCD8-GFP; UAS-mCD8-GFP/+; UAS-mCD8-GFP/+). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bar: 15 μm. Corresponds to Fig. 7m.
Live imaging of lateral pentascolopidial (Lch5) organ (green) in L3 larva expressing mCD8-GFP (green) to mark cell membranes: elav>mCD8-GFP (w1118, elave-Gal4/UAS-mCD8-GFP; UAS-mCD8-GFP/+; UAS-mCD8-GFP/+). The Lch5 organ is a particular chordotonal organ, which plays a role by proprioceptive locomotion control. Each frame is a single plane. Scale bar: 8 μm.
Live imaging of midgut enterocytes cells (green) in the L3 larva expressing NP1>GFP-Atg8a; foxo (w1118; NP1-Gal4/UAS-GFP-Atg8a; UAS-foxo/+). Overexpression of FOXO in the midgut enterocytes induces autophagy (green dots are autophagosomes). The fine-focus was changed during the live imaging to image through different planes. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7n.
Live imaging of midgut interstitial cells (green) in the L3 larva expressing NP1>GFP-Atg8a; foxo (w1118; NP1-Gal4/UAS-GFP-Atg8a; UAS-foxo/+). Overexpression of FOXO induces autophagy in the midgut interstitial cells (green dots are autophagosomes). The fine-focus was changed during the live imaging to image through different planes. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7o.
Live imaging of haemocytes in the L3 larva expressing Hml>dsRed (magenta, in the first movie) to mark the entire cytoplasm of the haemocytes and Jupiter (green, in the second movie) to mark the microtubules of the haemocytes: Hml>dsRed (w1118; Hml-Gal4/+; UAS-dsRed/+) and Jupiter-GFP trap (w1118; +; Jupiter-GFP). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 7p-q.
First movie shows live imaging of dorsal muscles (green) in L3 larva expressing MHC>GFP-Atg8a; foxo (w1118; MHC-Gal4/UAS-GFP-Atg8a; UAS-foxo/+), in which high level of FOXO induces autophagy (green dots are autophagosomes). Second movie shoes live imaging of dorsal muscles (green) in L3 larva expressing Nrg-GFP t
Live imaging of single-cell (first movie) and multi-cell (second movie) laser wounding in the dorsal epidermis of L3 larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) to mark cell membrane and nuclei in the epidermis: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 10.
Redistribution of PIP3 reporter, tGPH (green) and FOXO (magenta) shuttling after wounding in the cells directly surrounding the wound. Wound healing was performed in the dorsal epidermis of L3 larva expressing tGPH (tubulin:GFP-PH) and foxo-mCherry (w-; tGPH; endo-dfoxo-v3-mCherry). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 11a.
Lowering of insulin receptor signalling in the larvae by expressing a dominant negative version of the insulin receptor (InRDN), reduced accumulation of the PIP3-reporter tGPH (back) at the wound edges and slows down the wound healing processes. Transgene genotypes: control (w1118; tGPH/+; A58-Gal4/+) and A58>InRDN (w1118; tGPH/+; A58-Gal4/UAS- InRDN). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 11b.
Supplementary Video 15 | Long exposure to anesthetic increases larval lethality during live imaging.
The early L3 instar larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) was exposed for 6 min instead of 3.5 min to diethyl ether. A single-cell laser wound was made in the dorsal epidermis. The larva died during the experiment. Transgene genotypes of larvae: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Projections of a time-lapse series. Each frame is merged from 57 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 9g.
Example of a larva that did not remain immobile for the entire experiment. The reason for the larva waking up could be that (i) the exposure to diethyl ether was to short or (ii) the larvae in larval cage were too crowed or (iii) they were not completely dried (Table 5, Step 17). A single-cell laser wound was produced in the dorsal epidermis of a larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) and live imaged. Transgene genotypes of larvae: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Projections of a time-lapse series in early L3 larvae. Each frame is merged from 57 planes spaced 0.28 μm apart. Scale bars: 20 μm.
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Kakanj, P., Eming, S.A., Partridge, L. et al. Long-term in vivo imaging of Drosophila larvae. Nat Protoc 15, 1158–1187 (2020). https://doi.org/10.1038/s41596-019-0282-z