Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg

Begin with a confocal microscope setup containing a fixed, genetically modified Drosophila leg mounted within the space between two coverslips. The motor neuron axons in the leg express a membrane-tagged fluorescent protein.

Use a single laser and two detectors to capture fluorescent signals from the motor neuron axons and background autofluorescence of the cuticle which is the outermost layer of the leg.

Adjust imaging settings for optimal resolution and image quality.

Fine-tune the brightness to ensure a bright axonal signal and a saturated cuticle autofluorescence.

Capture an image with both axonal signal and cuticle autofluorescence.

Using appropriate image processing software, open the captured image.

Split the channels and subtract the background cuticle autofluorescence from the axonal signal.

Adjust the brightness and contrast of both signals to improve image clarity.

Merge the channels to generate a merged image to visualize the motor neuron axons within the leg segment.