The overall goal of this protocol is to allow qualitative and quantitative assessment of motor neuron axon arborization patterns using light sheet microscope and image analysis software on mouse embryos. This method can help to understand motor neuron development in coordination with complex movement. The main advantage of this technique is that you can observe and image axon arborization patterns of motor neurons from various angles and quantitatively analyze each individual nerve.
Demonstrating the procedure will be Ya-Ping Yen and Ee Shan Liau, two graduate students from my laboratory. To begin this procedure, fix the embryos individually in a 24-well plate with one milliliter of freshly prepared 4%paraformaldehyde in PBS per well at four degrees Celsius on a shaker overnight. The next day, wash the fixed embryos at least three times, each for five to 10 minutes, with one milliliter of 1X PBS and incubate them overnight at four degrees Celsius.
Next, permeabilize each embryo in one milliliter of 0.5%PBST overnight at four degrees Celsius on a shaker. Block each sample with one milliliter of 10%FBS prepared in 1X PBS overnight at 4 degrees Celsius on a shaker. Subsequently, incubate each embryo with one milliliter of anti-GFP primary antibody at 4 degrees Celsius for 72 hours with constant shaking.
After the primary antibody incubation, wash each embryo with one milliliter of 0.5%PBST over the course of one to two days at four degrees Celsius on a shaker. Afterward, apply one milliliter of secondary antibody and incubate overnight in the dark at four degrees Celsius with constant shaking. The following day, wash each embryo with one milliliter of 0.5%PBST at least three times at four degrees Celsius on a shaker over the course of two to three days.
Then, incubate each embryo in a commercial clearing reagent with a refractive index of 1.49 nd in a 1.5 milliliter tube protected from light at room temperature overnight. 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 X, Y, 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 lightpath parameters such as Detective Objectives, Laser Blocking Filter, Beam Splitter, Cameras, and Lasers.
Check the Pivot Scan check box for shadow reduction. Then, define the acquisition settings, Bit Bepth to 16-bit, Zoom to the range between 0.36 and 0.7X, single site illumination or dual site 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 lightpath to allow detailed acquisition of images of fine arborized structures on individual motor nerves. For multi-dimensional 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 czi format.
To quantify axon arborization, open the image file in the imagining 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 Autopath 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. LSFM provides detailed 3D visualization of motor axon arborization in mouse embryos.
Motor neurons are subjected to the above-described protocol and labeled with transgenically-expressed GFP. To image axon arborization in greater detail and to perform quantification, magnification can be adjusted so that every finely arborized structure can be revealed. Finally, axon arborization pattern of individual forelimb nerves can be reconstructed using image analysis software.
The autopath algorithm in the software traces filament and quantifies the total number of axon terminals. After watching this video, you should have a good understanding of how to prepare samples and image motor neuron axons of mouse embryos using light sheet fluorescense microscope. Also, you will know how to quantitatively assess the arborization pattern of each individual motor nerves by MRS.
