A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

J Neurogenet. 2021 Sep;35(3):306-319. doi: 10.1080/01677063.2021.1892096. Epub 2021 Mar 10.


Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Keywords: APL neuron; IR76b; Tissue clearing; ethyl cinnamate; mushroom body; near-infrared fluorescent protein.

MeSH terms

  • Animals
  • Animals, Genetically Modified*
  • Drosophila
  • Green Fluorescent Proteins
  • Imaging, Three-Dimensional / methods*
  • Larva
  • Microscopy, Confocal / methods
  • Optical Imaging / methods*
  • Transgenes


  • Green Fluorescent Proteins