Visualization of synaptic markers in the optic neuropils of Drosophila using a new constrained deconvolution method

The fruitfly Drosophila melanogaster offers compelling genetic advantages for the analysis of its nervous system, but cell size precludes immunocytochemical analysis of wild-type structure and mutant phenotypes beyond the level of neuronal arborizations. For many antibodies, especially when immunoelectron microscopy is not feasible, it would therefore be desirable to extend the resolution limit of confocal microscopy as far as possible. Because high-resolution confocal microscopy suffers from considerable blurring, so-called deconvolution algorithms are needed to remove, at least partially, the blur introduced by the microscope and by the specimen itself. Here, we present the establishment and application of a new deconvolution method to visualize synaptic markers in Drosophila optic neuropils at the resolution limit of light. We ascertained all necessary parameters experimentally and verified them by deconvolving injected fluorescent microspheres in immunostained optic lobe tissue. The resulting deconvolution method was used to analyze colocalization between the synaptic vesicle marker neuronal synaptobrevin and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of light, which could not be distinguished without deconvolution.