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

Abstract

Source: Liau, E. S. et al. Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy. J. Vis. Exp. (2018).

This video demonstrates the use of light sheet fluorescence microscopy to image motor neuron projections and axon branching in transgenic mouse embryos. It outlines the steps involved in sample preparation, imaging, and 3D reconstruction of axonal branching for assessing motor neuron growth and connectivity.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Light sheet fluorescence microscopy (LSFM) (2–3 h)

1. Sample set-up
   1. Set up 5X/0.1 illumination optics and 5X/0.16 detection optics. Assemble the sample holder and sample capillary (1.5 mm internal diameter, green) and place them into the microscope.
      NOTE: Refer to the microscope's Operating Manual for detailed set-up procedures.
   2. Mount the embryo as follows:
      1. Prepare the P200 pipette tip by cutting away the upper part so that it fits the diameter of the capillary and remove the pointed part (~ 3 mm) for the sample attachment.
      2. Melt the blunted end of the pipette tip using a small flame.
      3. Extinguish the flame and quickly attach the embryo vertically onto the melted end.
      4. Fit the upper part of the pipette tip with the sample capillary.
   3. Allow the chamber buffer to equilibrate with the embryo for 1 min to clear off debris or bubbles.
   4. Using the imaging software, select Locate capillary under the Locate tab and adjust the x, y, and z axes to position the sample capillary.
   5. Select Locate sample and zoom to 0.6X to focus on the embryo. Rotate the embryo to ensure that the long axis of the imaged limb aligns with the light path of the two light sources (Figure 1B).
2. Image acquisition
   1. Under the Acquisition tab, define the light path parameters such as detection objectives, laser blocking filter, beam splitter, cameras, and lasers.
      NOTE: The green fluorescent protein (GFP) channel is used in this experiment (Excitation wavelength: 488 nm; emission filter: bandpass (BP) 505 to 545 nm; beam splitter: SPS LP 560).
   2. Check the pivot scan checkbox for shadow reduction.
   3. Define the acquisition settings: bit depth, 16 bit; zoom, 0.36-0.7X; single-side illumination (left/right) or dual-side illumination.
   4. Click Continuous and set the laser intensity, exposure time, laser power, and light sheet position to acquire sharper images.
      NOTE: The laser power is kept as low as possible to minimize photodamage.
   5. Press STOP to end image acquisition.
3. Multidimensional acquisition
   1. Define the z-stack by moving the Z position for the first and last image. Click Optimal to set the slice number.
   2. Click Start Experiment to acquire the selected z-stack.
   3. When it is done, save the image in .czi format.
4. Image processing (optional)
   1. Proceed with Dual side fusion under the Lightsheet Processing channel if dual-side illumination is applied. Multiview processing is necessary if multi-views are acquired to combine images from multiple angles.
   2. Create a 2D image using data from the highest intensity pixels along the projection axis under the Maximum Intensity Projection channel.
   3. Under the Copy channel, click Subset to select subsets of images from the original set.

2. Quantification of Axon Arborization (30 Min for Each Individual Nerve)

1. Open the image file in the imaging analysis software (by default, the image containing the XYZ data is opened in Surpass mode and as a 3D-rendered view).
2. Adjust the image color, brightness, and contrast using the Display Adjustment window (Edit | Show Display Adjustment) to detect filaments based on local intensity contrast.
3. Click on the Add New Filaments icon and select the Autopath (no loop) algorithm in the drop-down menu.
4. Select the region of interest (in this case, the segmenting axon of interest). Click Next when finished.
5. Define the starting and seed points by assigning the largest and thinnest diameter measurements, which can be measured using the Slice mode.
6. 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 + right-click at the points of interest.
7. Select manual thresholds for seed points to ensure that all of the visible arborization is marked. Change the pointer mode (Navigate | Select) and then Shift + left-click at the points of interest to manually add or remove seed points.
   NOTE: It is important to manually remove background noises and signals from neighboring nerves.
8. Check the box Remove seed points around starting points.
9. Check Remove disconnected segments to allow the exclusion of points that are too far away, and that may represent background noise. Indicate the Maximum Gap Length in the next step to define the upper limit for exclusion.
10. Adjust the threshold for background subtraction, which uses a Gaussian filter to estimate the background intensity of every voxel.
11. Set the automatic threshold for dendrite diameter using the Approximate cross-section area algorithm.
12. Skip the steps for spine calculation.
13. Finish the process and choose the desired style and color.
14. Uncheck the volume box in Properties to view only the reconstructed axons.
15. Under the Statistics tab, select Detailed. Use Filament No. Dendrite Terminal Points to quantify the motor nerve terminals as an indicator of motor axon arborization.
    NOTE: Statistical annotation can be added if desired.
16. Export the image of the reconstructed axon as a .tif file.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hb9::GFP	The Jackson labortory	005029	Collect embryos of embryonic day 13.5 (E13.5)
4% Paraformaldehyde (PFA)			For 200ml: Add 20ml 10X PBS, 8g PFA in double-distilled water. Adjust pH to 7.4 with NaOH (10N). Filter sterilize and store at -20 °C.
Phosphate buffer saline 10X (PBS 10X)			For 1L: Add 80 g NaCl, 2 g KCl,14.4g disodium hydrogen phosphate, 2.4 g potassium dihydrogen phosphate and top up with double-distilled water. Autoclave and store at RT.
Triton X-100	Sigma	X100-500ML
Fetal Bovine Serum	ThermoFisher	26140079
Sheep polyclonal anti-GFP	AbD Serotec	4745-1051	1:1000
Alexa Fluor 488 donkey anti-sheep	Invitrogen	A-11015	1:1000
RapiClear 1.47 clearing reagent	SunJin Lab	RC147001
1.5ml micro tube	Sarstedt	72.690.001
24 wells plate	ThermoFisher	142475
5 SA Tweezer	ideal-tek	3480641
Iris Scissors striaght sharp/sharp	Aesculap	BC110R
Microsurgery Scalpels, single use	Aesculap	BA365
Dissecting microscope	Nikon	SMZ800
Shaker	TKS	RS-01
Lightsheet Z.1 microscope	Carl Zeiss Microscopy
Imaris 8.4.0 image analysis software	Bitplane, Zurich, Switzerland
B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9::GFP mice)	The Jackson Laboratory	005029