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Take a chemically fixed and cleared tissue segment from a transgenic mouse embryo expressing green fluorescent protein (GFP) in spinal motor neurons.
Mount the tissue vertically onto a modified pipette tip.
Attach the tip to a capillary secured within the light sheet fluorescence microscope.
Place a buffer-filled sample chamber beneath the tissue to immerse it and clear debris.
Using imaging software, align the tissue in the light path.
Begin image acquisition.
The light source emits a thin sheet of laser light, exciting GFPs in a single plane of the tissue to emit fluorescence.
A detector positioned perpendicular to the light path captures the emitted fluorescence.
Acquire images of the spinal motor neurons to generate a 3D reconstruction of the axonal branching morphology.
Use software to map axons, identifying branching patterns and endpoints to assess motor neuron growth and connectivity.
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