ilastik install and run for Syn Bot (Mac Version)

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0:00 - 1:00 Install ilastik by downloading it from ilastik.org/download and running the installer. To run ilastik after initial installation, right-click the application and click open. You will then be prompted to open the application even though it is from an unrecognized developer.

1:00 - 2:38 Generate training images by running SynBot on a set of images so that any preprocessing steps will be the same as a SynBot run.

2:38 - 3:15 Copy two-color training images to a training images folder

3:15 - 3:30 Make empty folder for holding red images and green images

3:30 - 3:50 Run extract_channels.ijm macro from the extra code folder on the SynBot GitHub repository

3:50 - 4:40 Select merged images folder, then red images folder, then green images folder

4:40 - 4:58 Open ilastik and start a new Pixel Classification project

4:58 - 5:09 Save the ilastik project le to a safe place where you can use it to threshold with SynBot

5:09 - 5:25 Click add new image and select the red training image (or images)

5:25 - 5:35 Click the "2. Feature Selection" drop down then "select features" and use Ctrl + A to select all features.

5:35 - 5:37 Click the "3. Training" drop down to advance

5:37 - 5:40 Zoom in image using Cmnd + +

5:40 - 6:05 Use the Label 1 to mark several examples of foreground puncta. Only a handful are needed to get started.

6:05 - 6:12 Click Label 2 and use this to label some examples of background regions.

6:12 - 6:21 Click Live Update to see a rendering of the ilastik model that will update when new markings are added. Use Cmnd + - to zoom out and check how well this model is working.

6:21 - 6:30 Save the ilastik project. This saved model will be used by SynBot and you do not need to change anything in the steps 4 or 5 in the ilastik pipeline.

6:30 - 8:01 Repeat steps 8-17 for the green channel image

8:01 - 8:43 Open and run SynBot.ijm. Move ilastik to your applications folder. FIJI may not have permission to access ilastik otherwise.

8:43 - 8:59 Enter your analysis parameters, including the ilastik thresholding method.

8:59 - 9:15 Select ilastik.app application location. On Mac this will typically be in a path like ~/Applications/ilastik-1.4.0.app

9:15 - 9:20 Select the ilastik project file for thresholding the red channel.

9:20 - 9:25 Select the ilastik project file for thresholding the green channel.

9:25 - 9:30 Enter the threshold of ilastik condence to use for the analysis. ilastik will give a condence score from 0 to 1 of how likely the given pixel is part of the foreground. A good threshold is something between 0.5 and 0.9.

9:30 - 10:30 View results