A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy

Area of Science:

• Cell Biology
• Microscopy
• Molecular Signaling

Background:

• Traditional cell segmentation is complex, requiring specialized equipment and computational methods.
• Monitoring intracellular signaling pathways like STAT signaling often demands sophisticated techniques.

Purpose of the Study:

• To develop a high-fidelity protocol for nucleus and cytoplasm segmentation in cell cultures.
• To apply this protocol for monitoring interferon-induced signal transducer and activator of transcription (STAT) signaling.
• To offer a cost-effective alternative to current segmentation and signaling monitoring methods.

Main Methods:

• Detailed guidelines for sample preparation, image acquisition, and segmentation using conventional techniques.
• Application of the protocol to monitor interferon-induced STAT signaling.
• Comparison with neural-network-based segmentation approaches.

Main Results:

• The developed protocol achieves high-fidelity nucleus and cytoplasm segmentation.
• Performance is comparable to neural-network-based segmentation methods.
• Enables simultaneous monitoring of two STAT isoforms using conventional equipment.

Conclusions:

• This protocol provides a reliable and cost-effective method for cell segmentation and STAT signaling analysis.
• It is adaptable for other nucleo-cytoplasmic shuttling molecules and high-throughput applications.
• Improves upon existing assays for STAT signaling monitoring.