PHANTAST FIJI plugin tutorial
FIJI (Fiji Is Just ImageJ) is a distribution of ImageJ containing additional libraries and plugins focused on life science research.
This page aims at getting you started with the PHANTAST plugin for FIJI. It contains detailed instructions as well as videos. If you still have questions, do not hesitate to let us know!
- Getting started
- Use of PHANTAST output
- Batch processing for confluency determination
This short tutorial will walk you through the installation of the plugin and how to process images. See the video below for a more detailed tutorial.
- Download the most recent version of FIJI for your operating system
- Download the PHANTAST plugin for FIJI
- Install the plugin by dragging and dropping the .jar files onto the FIJI command bar (for more information on plugin installation, see the FIJI documentation)
- Re-start FIJI or use the Help -> Refresh Menus command
- Open a phase contrast microscopy image
- Convert it to a 32-bits representation by using the Image -> Type -> 32-bit command
- Open PHANTAST by going in Plugins -> Segmentation -> PHANTAST
This procedure is demonstrated in the following video:
It is not practicable to process images one at a time if you are interested in determining culture confluency, which requires the processing of many images.
There are two approaches: using macros or using stacks of images. Both methods yield similar results (although using stacks can be faster if you have a multi-core system), so it is mostly down to your preference.
- Open the macro window by using the menu command Process -> Batch -> Macro
- Select the input folder containing the images to process
- Select the output folder where the processing outputs will be saved (if any)
- Sample macro that will process images with sigma = 1.2 and epislon 0.03, doing halo correction, computing confluency and outputting the binary segmentation mask:
run("32-bit");
run("PHANTAST", "sigma=1.20 epsilon=0.03 do compute new");
- The general form is:
run("32-bit");
run("PHANTAST", "args");
where args can be the following:
| Argument | Function |
|---|---|
| sigma=x | Set the parameter sigma to x |
| epsilon=y | Set the parameter epsilon to y |
| do | Do halo correction. If omitted, only local thresholding is applied |
| compute | Compute image confluency, results are saved in a Results Table |
| new | Save binary segmentation mask output in chosen output folder |
See video below for more information.
- Create a stack of images holding all the images you want to analyze. This is easily done by using the File -> Import -> Image sequence menu command. Be sure to check the box to convert the images to 8-bit grayscale.
- Convert the stack to 32-bit using the Image -> Type -> 32-bit menu command.
- Open PHANTAST in Plugins -> Segmentation -> PHANTAST
- Modify segmentation parameters as necessary
- Tick the "Compute confluency" checkbox
- Click on "OK" to start the analysis
- Click "YES" to confirm that you want to process all images in the stack
- The results will appear in a Results table. Those can be copied and pasted in your software of choice for further analysis
See video below for more information.
