This article details a method for imaging Drosophila brain neurons using confocal microscopy. The focus is on visualizing and tracing the organization of medulla neurons and their synaptic connections.
Begin with a glycerol-cleared Drosophila brain in antifade mounting media.
The brain contains medulla neurons arranged in layers, which organize synaptic connections, and columns, which coordinate neural circuits.
A few of these neurons are labeled with green fluorescent markers, contrasting with the red fluorescent photoreceptor axons.
Position the brain ventral side up. Place a coverslip with small clay patches on the sides to prevent crushing the brain sample.
Under a microscope, obtain the horizontal view image stack, containing the green-labeled neuron.
Remove the coverslip and reposition the brain, anterior side up.
Capture an image stack of the frontal view of the same neuron.
Using the software, combine these images, remove the photoreceptor channel, and visualize the 3D image of the neuron.
Trace the axon and then the dendrites, following each branch to its termination, to map dendritic organization across medulla layers and columns.
To mount the brain in the horizontal orientation, transfer a glycerol-cleared brain into a 20-microliter drop of anti-fade mounting medium, deposited centrally on the microscope slide. Then, on a coverslip, attach small patches of clay at the four corners to prevent the coverslip from crushing the brain.
Now, under a dissecting microscope, position the brains in the ventral up position. Use the convex dorsal surface of the brain as a landmark to identify the orientation of the brain sample. This will provide a horizontal view of the neuron. Then attach the coverslip.
Using a confocal microscope, ideally equipped with GaAsP detectors. Obtain the horizontal view image stack with a high numerical aperture objective and a digital zoom of 2.5. Acquire at least 180 optical sections, have 512 square pixels, and a step size of 0.2 microns.
After taking the first image stack, remove the coverslip from the slide and reposition the brain in the anterior up position, which provides a frontal view. Then, using the same technique, make a new image stack of the frontal view of the same neurons imaged in the horizontal view. Be sure to also record the location of the neuron of interest with respect to the medulla neuropil.
Identifying the same neurons is feasible under lower magnification. However, if signal is lost because the tissue is deep, just image the ventral half of the brain.
The goal of this procedure is to trace neurites and to assign reference points for registration. First, open the recombined image file. Then go to Edit, then Show Display Adjustment, and turn off the photoreceptor channel, which is red.
Next, visualize the image in the surpass mode. If the computer is equipped with the stereograph system, turn on stereo and use the Quad Buffer mode to visualize 3D images. Add new filaments. Go to Surpass, then Filaments. And click on the tab labeled "Skip automatic creation, edit manually." Then click on the Draw tab and select AutoDepth.
Next, select Settings. Toggle the Line option and input the appropriate pixel number for better visualization. Then toggle Show Dendrites, Beginning Point, and Branching Point, and set the Render Quality to 100%. Now select the Draw tab and start tracing neurites. Start with the axon, then move to the dendrites. The axon and dendrites of the transmedulla neurons should be easy to differentiate.
After tracing the neurites, go back to Settings and detoggle both Beginning Point and Branching Point. Then go to Surpass, then Export. Select objects and save the filament as an Inventor file.
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