Colocalization
The Colocalization command identifies objects from two or more input classes that overlap each other. Overlapping objects are defined as colocalising. EVAnalyzer assigns all colocalising objects a shared tracking ID, allowing them to be grouped in the results table.
Parameters
| Parameter | Description |
|---|---|
| Classes to colocalize | Two or more object classes that must all have an overlapping member for an object to be considered colocalising |
| Filter classes | Additional classes that must also overlap (optional secondary filter) |
| Class for overlapping areas | A class assigned to a new object representing the actual overlap area |
| Allow multi-object colocalization | If enabled, an object may colocalize with more than one partner from the same class |
| Min colocalization area | Minimum overlap area in the selected unit |
| Size unit | Unit for the min area threshold |
Colocalizing handling
When an object is found to colocalize, one of two operations is applied:
- Move — the object’s class is changed to a new class. The Object ID is unchanged.
- Copy — a new object is created with the new class; the origin object’s ID is recorded in the copy’s origin object ID field.
A new colocalizing area object is created for the overlapping region. This object is assigned to the class specified by Class for overlapping areas and can be used for further measurements.
All colocalising objects — input objects and the colocalization-area object — receive the same tracking ID. In the results table, rows with the same tracking ID appear adjacent to each other.
Example: Two-channel spot colocalization
- Classify spots in Cy5 channel →
cy5@spot - Classify spots in Cy7 channel →
cy7@spot - Add a Colocalization step:
- Classes:
cy5@spot,cy7@spot - Class for overlapping areas:
coloc@cy5cy7
- Classes:
- All Cy5 spots that overlap with a Cy7 spot (and vice versa) receive a common tracking ID.