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.

To image the legs, use the 488-nanometer laser and two detectors simultaneously to set up the first track for obtaining both the GFP and cuticle autofluorescence. Select a 20 to 25x oil immersion objective and set the resolution to 1024 by 1024 pixels, with a 12-bit depth. And set the z spacing to 1 micrometer. Load the slide onto the microscope stage.

And using the same laser power for both detectors, adjust the gain of the first detector to obtain a bright GFP signal. And adjust the second detector to ensure that some areas with a high cuticle signal produce a saturated signal in this detector. Then, image the legs, using the tile or position options to capture the entire leg, if a leg is extended, or too large to be imaged in a single frame.

For image processing, open the confocal stack in ImageJ Fiji. And use the formats plugin to open any images that are not in TIFF format. To split the channels, select Image, Color, and Split Channels. To subtract the cuticle signal from the GFP signal, open Process and Image Calculator, and select the stack from detector one as Image1.

In the operation window, select Subtract, and select the track from Detector 2 as Image2, so that only the endogenous GFP signal will be obtained. Use Image, Stacks, Z Protect to generate max intensity projection for the endogenous GFP signal. Use the controls in the brightness control window to adjust the brightness and contrast.