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

The fruitfly Drosophila melanogaster offers compelling genetic advantages f or the analysis of its nervous system, but cell size precludes immunocytoch emical analysis of wild-type structure and mutant phenotypes beyond the lev el of neuronal arborizations. For many antibodies, especially when immunoel ectron microscopy is not feasible, it would therefore be desirable to exten d the resolution limit of confocal microscopy as far as possible. Because h igh-resolution confocal microscopy suffers from considerable blurring, so-c alled deconvolution algorithms are needed to remove, at least partially, th e blur introduced by the microscope and by the specimen itself. Here, we pr esent the establishment and application of a new deconvolution method to vi sualize synaptic markers in Drosophila optic neuropils at the resolution li mit of light. We ascertained all necessary parameters experimentally and ve rified them by deconvolving injected fluorescent microspheres in immunostai ned optic lobe tissue. The resulting deconvolution method was used to analy ze colocalization between the synaptic vesicle marker neuronal synaptobrevi n and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of lig ht, which could not be distinguished without deconvolution. J. Comp. Neurol . 429: 277-288, 2001. (C) 2001 Wiley-Liss, Inc.