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.
Highlights
IntroductionHigh-resolution 3D reconstructions with isotropic resolution from entire green fluorescent protein (GFP)-expressing flies are obtained by applying image fusion from orthogonal directions
The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level
By combining our innovations in clearing technique, light-sheet imaging, and computational multi-view combining, we show for the first time the systemic 3D imaging of endogenous fluorescent markers in intact D. melanogaster for multiple developmental stages and adult flies with isotropic spatial resolution and long-term signal stability
Summary
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. Available mounting media, such as DPX, Glycerol, 2,2′-Thiodiethanol (TDE)[29], VECTASHIELD®, FocusClear®, ProLongTMGold, RapidClear®, HistodenzTM or recently published direct immersion media such as FRUIT6, ClearT7, ScaleS8, SeeDB9, SeeDB210 and RTF11 have such RI matching properties Each of these solutions has some limitations regarding the preservation of morphology, fluorescent stability, viscosity, penetration depth and the capacity to render tissue transparent[8,10,11]. As previous studies have shown that the autofluorescence of optically cleared flies can be imaged with micrometre-resolution in a short period of time by lightsheet microscopy[36], an efficient tissue-clearing technique, which preserves the stability of endogenously expressed fluorescent proteins would be a key innovation to overcome current technological disadvantages
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