This example is similar to Measuring Mean Fluorescence Intensity on a Ring around the primary Object and also uses the same data. This example shows how to count the number of objects on a ring that is associated with the main object, e.g. the cell nucleus. The images are taken from AF568 stained nuclei. The mitochondria are stained with AF488. The channel of the nuclei is used for image segmentation. The ZOI-segmentation method attributes a zone of influence (ZOI) and a ring to each detected nucleus. This area is used as a search range to detect subobjects, in this case the mitochondria.
Counting the Number of Objects in a Ring around the Nucleus
Step 2: Frame
For more information, see Step 2: Frame.
Step 3: Automatic Segmentation
- For more information, see Step 3: Automatic Segmentation.
- Additionally, you need to set the segmentation parameters for the object Mitochondrion. Choose suitable parameters to segment the objects.
- You have detected the mitochondria on the ring.
Step 4: Region Filter
For more information, see Step 3: Automatic Segmentation.
See also
Step 5: Features
You can define individual measurement features for each class. The measurement features defined for one class you can copy to the other classes via Copy to all.
- Select the class for which you want to define measurement features, and click Edit. From the list of features on the right you can add features to the selected features list on the left.
- These features are automatically defined for every object during image analysis. All classes have ID of the parent and ID as default features. This allows you to later to group the associated parameters from the excel lists, if necessary.
- The class Ring additionally has Area and Count as default parameters.
- In this example, we count the number of objects (the number of mitochondria fragments) within each ring. To attribute the number of mitochondria fragments to the Primary Object, select Mitochondria and click Edit.
- The Feature Selection dialog opens.
- In the parameter list on the right, select the feature Count. In the Copy drop-down menu, click
.
- This measurement feature is copied to the corresponding Primary Object.
Step 6: Preview Result
In this step you see a preliminary result of the image analysis.
- Click on the different objects in the Analysis tab to get the preliminary measurement result for all objects.
- Select the Primary Object to get a list with preliminary measurement results with the features ID of the parent, ID, Area, and Ring Mitochondria Counts. Note that the results are preliminary and only include the part of the image you see in the viewport.
- The result of the image analysis shows Ring Mitochondria Counts as a feature of Primary Object.
- In the table, click on column Ring Mitochondria Counts to sort the entries in increasing or decreasing order.
- Click on Finish to save the analysis settings and to close the wizard.
You can now run the analysis as described in Measuring Mean Fluorescence Intensity on a Ring around the Primary Object.