JoVE Encyclopedia of Experiments
Neuroscience
0 views • 5:40 min • May 29th, 2025
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.
Set up 5X/0.1 illumination optics and 5X/0.16 detection optics. Assemble the sample holder and sample capillary. Prepare the sample chamber by filling it with clearing solution. To mount the embryo, prepare a P200 pipette tip by cutting away the upper part so that it fits the diameter of the capillary
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