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

Abstract

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.

Immunocytochemical localization of synaptic proteins to photoreceptor synapses of Drosophila melanogaster

“…Related studies on other synapses in Drosophila are mostly lacking. A previous confocal study reveals several synaptic epitopes in the terminals of the compound eye’s photoreceptors (Hiesinger et al, 2001), but such studies rarely attain a resolution sufficient to differentiate the subcellular localizations, and therefore little is known regarding the precise localization of proteins in the T-bar ribbon or associated synaptic organelles.…”

Quantitation of Contacts Among Sensory, Motor, and Serotonergic Neurons in the Pedal Ganglion of Aplysia

“…Recently, confocal microscopy has been used for high resolution analysis of connections between labeled neurons (Gan et al 1999;Cabirol-Pol et al 2000;Jacoby et al 2000;Hiesinger et al 2001;Hammar and Maxwell 2002;Gray and Weeks 2003). We used this technique to identify sites of proximity between sensory neurons and their followers.…”

Confocal Laser Scanning: of Instrument, Computer Processing, and Men

“…Even with an ideal pinhole size and a circular pinhole this already and inevitably leads with single monochromatic laser illumination to the addition of "rainbow" photons at the upper and lower faces of the focal plane. This is represented in confocal Z-images as a "flaring" instrument-measured PSF (Hiesinger et al, 2001). A square or rectangular pinhole adds additional small distortions of the instrument's PSF.…”