Visualizing Motor Neuron Projections and Axon Branching Using Light Sheet Fluorescence Microscopy

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, and remove the pointed part for the sample attachment.

Then, melt the blunted end of the pipette tip using a small flame. Extinguish the flame and quickly attach the embryo vertically onto the melted end. Fit the upper part of the pipette tip with the sample capillary, and put the prepared sample chamber into the microscope. Using the imaging software, select Locate Capillary under the Locate tab, and adjust the xy and z-axes.

Next, select Locate Sample and zoom to 0.6X to focus on the embryo. Allow the chamber buffer to equilibrate with the embryo for some time to clear off any debris or bubbles. For image acquisition under the Acquisition tab, define the light path parameters, such as detection objectives, laser blocking filter, beam splitter, cameras, and lasers.

Check the pivot scan checkbox for shadow reduction. Then, define the acquisition settings, bit depth to 16 bit. Zoom to the range between 0.36 and 0.7X single side illumination or dual-side illumination. Click Continuous, and set the laser intensity, exposure time, laser power, and light sheet position. Subsequently, press stop to end image acquisition.

It is important to ensure that the transparent sample is positioned within the shortest light path to allow detailed acquisition of images of fine upright structures on individual motor nerves.

For multidimensional acquisition, define the Z stack by moving the Z position for the first and last image. Click Optimal to set the slice number. Then, click Start Experiment to acquire the selected Z stack. When it is done, save the image in dot CZI format.

To quantify axon arborization, open the image file in the imaging analysis software. Adjust the image color, brightness, and contrast using the display adjustment window to detect filaments based on local intensity contrast. Next, click on the Add New Filaments icon, and select the auto path algorithm in the dropdown menu. Select the region of interest.

Then, define the starting and seed points by assigning the largest and thinnest diameter measurements, which can be measured using the slice mode. Assign a starting point at the edge of the region of interest. To achieve manual addition or removal of starting points, first, change the pointer mode, Navigate Select, and then shift and right-click at the points of interest.

Next, select Manual Thresholds For Seed Points to ensure that all of the visible arborization is marked. Then, manually add or remove seed points. Check the box, Remove Disconnected Segments, and remove seed points around starting points. Subsequently, adjust the Threshold For Background Subtraction and finish the process.

It is necessary to remove background artifacts and confirm the detector path reflect true axon arborization for accurate quantification.

At the end, choose the desired style and color. Under the Statistics tab, select detailed, and use filament number dendrite terminal points to quantify the motor nerve terminals as an indicator of motor axon arborization. Then, export the image of the reconstructed axon as a .TIFF file.