TITLE: Imaris Coloc Protocol
AUTHORS: Hong-Yuan  Chu

[DESCRIPTION]
This protocol details the procedure of the imaris coloc protocol.

[STEPS]
SECTION: Background Subtraction
1. Open confocal files (.ND2) in ImageJ.
SECTION: Background Subtraction
2. Perform background subtraction on z-stack of images (process -> subtract background).
SECTION: Background Subtraction
2.1. Rolling ball radius: 50.0 pixels.
SECTION: Background Subtraction
3. Save z-stack as a .TIFF file.
SECTION: Background Subtraction
4. To select the .TIFF files containing folder open Imaris and press the “Choose Folder” icon.
SECTION: Background Subtraction
5. Double click the desired .TIFF files and they will automatically convert to .IMS format in the Arena.
SECTION: Background Subtraction
6. Open the new .IMS file.
SECTION: Region of Interest using Surface Function
7. To create a region of interest, click on the blue “add new surface” button to initiate the surface creation.
SECTION: Region of Interest using Surface Function
8. In the first step, select “skip automatic creation, edit manually”.
SECTION: Region of Interest using Surface Function
9. Next, select the contour tab that is in the “Draw” setting.
SECTION: Region of Interest using Surface Function
10. Slide the slice position to the bottom of the image.
SECTION: Region of Interest using Surface Function
11. In the resolution settings, select manual and preserve features and increase size to highest possible option.
SECTION: Region of Interest using Surface Function
11.1. This will maintain the size and dimension of the surface throughout the image and is important to keep uniformity.
SECTION: Region of Interest using Surface Function
12. Change from the “Board” display to “Mode”.
SECTION: Region of Interest using Surface Function
13. Select the “Circle” option under “Drawing Mode”, and set the parameters to the following: radius = 15 µm; number of vertices = 30.
SECTION: Region of Interest using Surface Function
14. Select the “draw” button in the bottom right corner While on the bottom slice.
SECTION: Region of Interest using Surface Function
14.1. The pointer must be set to “Select” and will not work under the “Navigate” option.
SECTION: Region of Interest using Surface Function
14.2. This can be changed by pressing the “Esc” button on the keyboard, or in the top right corner of the Imaris Arena.
SECTION: Region of Interest using Surface Function
15. Place the circle over the desired region in the image.
SECTION: Region of Interest using Surface Function
16. Change back to the “Board” display and under “Selection” press “Copy”.
SECTION: Region of Interest using Surface Function
17. Move the slice position to the top of the image and press “Paste”.
SECTION: Region of Interest using Surface Function
18. Press the “Create Surface” button to generate the surface construction.
 

SECTION: Region of Interest using Surface Function
19. To mask each channel based on the generated surface, click on the “Edit” tab and under mask properties select “Mask All”.
SECTION: Region of Interest using Surface Function
20. A pop up will appear and select the channel to mask.
SECTION: Region of Interest using Surface Function
20.1. Set voxels outside surface to: 0.00.
SECTION: Region of Interest using Surface Function
21. A new channel will be created that only contains image data within the region of interest created (where the surface was created).
SECTION: Region of Interest using Surface Function
22. Repeat steps 19 & 20 for each channel necessary.
SECTION: Region of Interest using Surface Function
23. Open “Display Adjustments” in the top “Edit” button and turn off the original channels so just the masked channels are on.
SECTION: Colocalization
24. Press the “Coloc” button to begin colocalization.
SECTION: Colocalization
25. Set Channel A to the first masked channel (either vGluT1 or vGluT2).
SECTION: Colocalization
26. Set Channel B to the other masked channel (aSyn) that is being measured against Channel A.
SECTION: Colocalization
27. Set the selection mode to threshold and use the automatic thresholding function (“Calculate Thresholds”) to set thresholding parameters.
SECTION: Colocalization
28. Click “Build Coloc Channel” to save the results as a new channel.
SECTION: Colocalization
29. Repeat steps 25-28 for each combination of channels that are needed (example: vGluT1/aSyn & vGluT2/aSyn).
SECTION: Colocalization
30. To export colocalization results, go to “Image Properties” under the edit tab and select the channel equating to the colocalization channel.
SECTION: Colocalization
31. Press the export button to export all colocalization results and repeat with each necessary channel.