Experimenters' guide to colocalization studies: finding a way through indicators and quantifiers, in practice.

Area of Science:

• Cell Biology
• Microscopy
• Image Analysis

Background:

• Multicolor fluorescence microscopy is crucial for studying subcellular component interactions.
• Colocalization analysis, assessing spatial coincidence of markers, is a key first step.
• Current methods are limited by optical resolution and lack clear guidance.

Purpose of the Study:

• To review parameters for evaluating colocalization in fluorescence microscopy.
• To provide a guideline for selecting and applying colocalization methods.
• To aid researchers in interpreting spatial relationships of molecular actors.

Main Methods:

• Discussion of two parameter types: indicators and quantifiers.
• Explanation of how indicators measure signal coincidence on a scale.
• Explanation of how quantifiers provide absolute measurements.

Main Results:

• Indicators and quantifiers offer different perspectives on colocalization.
• Both approaches are applicable given images as intensity collections and object collections.
• Most software offers colocalization options, but guidance is scarce.

Conclusions:

• A clear understanding of indicators and quantifiers is essential for accurate colocalization.
• The review proposes a guideline for choosing and using colocalization methods.
• Proper application of these methods enhances the investigation of molecular interactions.