StackMeasure Help
What is Stack Measure
StackMeasure is a plugin for ImageJ which can be used to identify features in one or more image stacks. It can then be used to measure the distances between those features in a number of different ways.
Installing
To install StackMeasure you simply need to unzip the install file into the plugins folder of your ImageJ installation. However, because the program uses the Java3D extensions to generate the output window you will also need to install these if you don't have them already. If you're using a Mac these are usually installed by default. If you're using windows you'll have to add them yourself by going to the Java3D download site and downloading and running the installer.
If you're using a version of ImageJ which has a Java environment bundled with it you'll need to use the Zip binaries (Expert mode) from the above link and unzip these into the lib folder of your ImageJ JRE. [Note haven't checked this is right yet - but the installer definitely misses the ImageJ custom JRE]
If the plugin works initially but dies when it get to "creating composite image" then this is often an indication that the Java3D classes haven't been found.
Running
To launch StackMeasure simply select Plugins > StackMeasure > StackMeasure from your ImageJ menu. You don't need to have opened your images before you launch the plugin.
Loading files
Stack measure works on one or more image stacks. Stacks can be TIFFs or LSM files. StackMeasure works with either RGB or BW files. If you use an RGB file the program will automatically segregate features based on their colour. If you're using BW files each file will be assumed to be a separate channel and will be assigned a pseudo-colour.
Setting preferences
Min Intensity
How bright does a voxel have to be to be considered part of a feature. Needs to be set high enough to exclude most background voxels. Probably best to start this setting high (50-60) and work down only if you aren't seeing your features. Setting this too low will make the plugin very slow as it has to assign lots of voxels, and may well end up eating all of your memory.
Hue flexibility
Only applies when using RGB files. Says how close in colour 2 voxels need to be to be considered part of the same feature. Set this higher if single features are being separated because of colour drift.
Min pixels per group
Says how small the smallest feature can be. Useful for removing small isolated clusters of voxels which are just noise. Increasing this will clean up your data and will make the plugin use less memory.
Distance between pixels/stacks
This is used to convert the virtual distance in the image into a real distance in microns. Provide the distance in µm between the centre of 2 adjacent pixels in the same stack for the first value, and the same pixel in 2 adjacent stacks for the second value.
Results
The results windows are split into 4 areas.
- The features list: This is on the left and details information about each identified feature. You can click on a feature to highlight it in the 3D view (in yellow) and in the composite image
- The 3D view: This is on the bottom right. It's a rotatable 3D view of the identified features. To move this view simply click and drag the mouse in the window. You can change the selected slice by scrolling your mouse wheel if you have one.
- The composite image: This is a separate ImageJ window. It interacts with the other windows to highlight selected features. If you scroll through the stack in this window you should see the selected stack slice move in the 3D view.
- The distance matrix: This is on the top right and show a matrix of measurements between features, with the distances measured in microns. By default it will show only the closest distance between the closest features of each colour and will measure from the centre of each feature. If you want to measure from the edges of features, or if you want to include all spatial features in the view then you can do this by changing the options under the "Measures" menu item.
If you want to save the list of spatial features, the distance matrix or the composite image stack you can do this from the File menu. For the tables you can choose to save either as text or HTML.
Problems / Bugs
StackMeasure is not yet finished and there are probably still bugs in it. If you're having problems with it please tell Simon Andrews and he'll do his best to sort them out.