Spot Colocalization

This tutorial demonstrates how to detect spots in two fluorescence channels and identify colocalising spot pairs - objects from both channels that overlap in the same spatial location.

Prerequisites

• Multi-channel images with spots in at least two distinct channels (e.g. Cy5 and Cy7).

Step 1: Define Classes

Class	Purpose
cy5@spot	Spots in channel 1
cy7@spot	Spots in channel 2
coloc@cy5cy7	Colocalisation overlap area

Step 2: Per-Channel Detection Pipelines

Create one pipeline per channel following the Spot Count workflow. Each pipeline should end with a Classify ROIs step that assigns the respective class:

• Pipeline 1 → cy5@spot
• Pipeline 2 → cy7@spot

Step 3: Colocalization Pipeline

Create a third pipeline with Empty input (no image channel needed). Add a single step:

Colocalization

Setting	Value
Classes to colocalize	cy5@spot, cy7@spot
Class for overlapping areas	coloc@cy5cy7
Min colocalization area	1 px²
Allow multi-object colocalization	disabled (one-to-one matching)

Step 4: Run and Inspect

After running:

• cy5@spot objects that colocalize are assigned a tracking ID shared with their matching cy7@spot partner.
• In the results table, sort by Tracking ID to see matched pairs side by side.
• The coloc@cy5cy7 class contains one object per colocalising pair, representing the overlap area.

Interpreting Results

Metric	Meaning
cy5@spot Count	Total Cy5 spots detected
cy7@spot Count	Total Cy7 spots detected
coloc@cy5cy7 Count	Number of colocalising spot pairs
Colocalisation %	coloc count / cy5 count × 100 (compute in export)

Filtering by colocalization area

Add a Classify ROIs step after Colocalization targeting coloc@cy5cy7 to filter out low-overlap events:

Filter	Value
Min area	5 px² (discard single-pixel overlaps due to border artefacts)