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Updated version of the protocol
Submit responseBecause of some changes in recent versions of ImageJ, an update to the originally published protocol is necessary. This update also includes some additional clarifications.
Image analysis and quantification are best performed in 3D (e.g., with the software Imaris by Bitplane), but can also be performed by the simpler method for 2D analysis using the free software ImageJ that is presented in this protocol. The intention of this protocol is to propose ideas of how such an analysis could be performed with ImageJ, rather than providing a stiff protocol, where the steps have to be followed without modifications. Please note that ImageJ constantly gets updated. The function of plugins can change with an update. The method as described here works for sure with ImageJ versions 1.42q-1.45g.
Step 2: If you open 16-bit files check "Autoscale".
Step 9: If the brightest pixel in your stack is lower than 255, use the intensity value of this brightest pixel instead of 255.
Step 11: Write down the value used for the lower threshold.
Step 14. Part A.
Segment the remaining fused synapses in the overlay images. It is recommended to perform this step semiautomatically via the command Find Maxima. Here the protocol branches off, depending on which version of ImageJ you use.
- ImageJ v1.43j and older: Process/Binary/Find Maxima..., Output type: Segmented Particles. Choose an appropriate value for "Noise Tolerance".
- ImageJ v1.43k and newer: Set the lower threshold of each segmented image to a gray value of 1, the upper threshold to 255, and do not press apply. (Image/Adjust/Threshold..., Lower Threshold Level: 1, Upper Threshold Level: 255, do not press apply!).
The location of the function Find Maxima has also been changed:
- ImageJ v1.43r and older: Process/Binary/Find Maxima...
- ImageJ v1.43s and newer: Process/Find Maxima..., Output type: Segmented Particles, check "Above lower threshold". Choose an appropriate value for "Noise Tolerance".
Step 14. Part B. Repeat Steps 12 and 13: Create overlays of the masks you created during the last steps with the images you saved in Step 10.
Step 14. Part C. Most probably it will not be possible to separate all synapses by using "Find Maxima". Therefore consider to continue the segmentation manually, as described in Step 14 of the original protocol.
Conflict of Interest:
None declared
