High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster

Abstract

The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applying image fusion from orthogonal directions. This methodological integration of novel chemical, optical, and computational techniques allows a major advance in the analysis of global neural circuit organisation.

Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure

“…Despite being a long-established model organism, Drosophila melanogaster has remained difficult to clear. Previous efforts in clearing Drosophila were either dependent on BABB (McGurk et al, 2007) or on a highly modified CUBIC approach (Pende et al, 2018). These methods are either laborious or dependent on antibody labelling.…”

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue

“…Especially for sample features that are highly absorbing and therefore cast shadows-such as the melaninrich eyes-multidirectional acquisitions can allow "filling in" of missing information. In addition, multidirectional datasets can be fused to achieve more isotropic resolution, a technique common in developmental light-sheet microscopy 74 that has recently been applied to cleared samples 75 . As the sample was mounted in a quadratic cuvette, we recorded overview stacks at 90° intervals to avoid imaging through the corners of the cuvette (Supplementary Figure 25).…”

“…were deeply anaesthetized with intraperitoneal injection of a mixture of 150 µl Ketamine (Ketalar, Bayer AG),75 µl Xylazine (Rompun, Parke-Davis) and 75 µl sterile water. When mice seized to breath and no toe reflex was present, mice were transcardially perfused with 20 ml ice cold phosphate buffered saline (PBS) after which 20 ml ice cold hydrogel monomer-fixative (4% acrylamide, 1% paraformaldehyde, 0.05% bisacrylamide, 1% VA-044 initiator in phosphate buffered saline)61 was infused.…”

Advances in Ex Situ Tissue Optical Clearing

“…Such system granted generation of thin and stable light sheet over >10 mm (Figure d) and much improved optical sectioning performance due to the presence of aspherical optical elements reducing aberration (Figure b). Very recently, light sheet generation was again optimized by Dodt's group to reduce the thickness of beam (thus enhance isotropy of imaging). Its performance was tested on fully cleared, GFP‐harboring D. melanogaster (for the first time efficiently depigmented with new solution based on CUBIC and containing 2,2′,2″,2 ‴ (ethylenedinitrilo)‐tetraethanol) by multi‐view imaging at orthogonal positions.…”