TITLE: Active zone protein clusters and dopamine axons in striatal slices
AUTHORS: Chuyu Chen

[DESCRIPTION]
This protocol detail the procedures for measuring the size and density of active zone proteins and axon terminals with Imaris software. 

[STEPS]
SECTION: Visualization of active zoon proteins and dopamine axons
1. Stain and amplify fluorescent labeled axons with GFP, cherry, and bassoon antibodies.
SECTION: Visualization of active zoon proteins and dopamine axons
2. Capture z-stack images with 100x oil lens (Nikon N-SIM Structured Illumination Super-Resolution Microscope), 4 μm (0.1 μm steps) 
SECTION: Visualization of active zoon proteins and dopamine axons
3. 9 images per section and 5 sections per mouse are used for analysis. 
SECTION: Quantification and colocalization analysis with Imaris 10.0
4. Open the 3D-reconstructed .nd2 files and convert to .ims files
SECTION: Quantification and colocalization analysis with Imaris 10.0
5. Initiate the surface creation with surface module
SECTION: Quantification and colocalization analysis with Imaris 10.0
6. Axon terminal
SECTION: Quantification and colocalization analysis with Imaris 10.0
6.1. Rename the surface module to GFP/mch.
SECTION: Quantification and colocalization analysis with Imaris 10.0
6.2. Select object-object statistics for algorithm settings.
SECTION: Quantification and colocalization analysis with Imaris 10.0
6.3. Select smooth and set the surface detail at 0.0643 μm. Background subtraction set the diameter of the largest sphere which fit into 0.421 μm
SECTION: Quantification and colocalization analysis with Imaris 10.0
6.4. Threshold set to auto for minimal value and none for max value, adject the value manually if needed. 
SECTION: Quantification and colocalization analysis with Imaris 10.0
6.5. Select “number of voxels of Img=1” for surface filter and set the min value to auto, adject the value manually if needed 
SECTION: Quantification and colocalization analysis with Imaris 10.0
7. Active zone protein
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.1. Select classify surfaces and object-object statistics for algorithm settings.
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.2. Select smooth and set the surface detail at 0.06 μm. Background subtraction set the diameter of the largest sphere which fit into 0.15 μm
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.3. Threshold set to auto for minimal value and none for max value, adject the value manually if needed. Select split touching objects with intensity based and enable seep points diameter set to 0.1 μm
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.4. Filter seed points with “Quality” and set the min and max value with auto.
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.5. Filter surfaces with “volume” and set the min at 0.003 μm^3 and max at 0.04 μm^3.
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.6. Filter colocalized active protein and axon terminal by select “overlapped volume ratio to surface surface=GFP/mch” and set the min=1  (100% volume of active zone protein overlapped with anxon).  
SECTION: Quantification and colocalization analysis with Imaris 10.0
8. Measure the axon length with filament module.
SECTION: Quantification and colocalization analysis with Imaris 10.0
8.1. Skip automatic creation, edit manually to 100% capture the axon surface.
SECTION: Quantification and colocalization analysis with Imaris 10.0
8.2. Select source channel to GFP or mcherry. And use “AutoPath” to trace the generated GFP/mch surface.
SECTION: Quantification and colocalization analysis with Imaris 10.0
7.7. Duplicate selection to new surface for statistic data of active zone protein within axon.
SECTION: Quantification and colocalization analysis with Imaris 10.0
9. Export all the statistical data to Excel for further analysis with GraphPad Prism9.