Multiplex live imaging approaches to interrogate the interplay of multiple signaling pathways

Area of Science:

• Cell Biology
• Biotechnology
• Cancer Research

Background:

• Multiplex live imaging visualizes multiple signaling pathways in real-time within living cells.
• It is essential for studying complex cellular networks, especially in cancer, due to signaling heterogeneity and dynamic changes.
• Conventional methods lack the temporal and single-cell resolution needed for dynamic signaling events.

Purpose of the Study:

• To review current strategies for multiplex live imaging.
• To highlight its applications in cancer signaling networks.
• To discuss advancements in biosensors, imaging, and computational analysis for exploring dynamic cellular regulations.

Main Methods:

• Spectral multiplexing using expanded fluorescent protein palettes and biosensor combinations.
• Intracellular, intercellular, and temporal multiplexing strategies.
• Orthogonal modalities like fluorescence anisotropy, lifetime, and Raman imaging.
• Computational approaches including spectral unmixing and deep learning algorithms.

Main Results:

• Enables simultaneous tracking of multiple signaling pathways within single cells.
• Reveals how diverse cellular inputs are integrated into cellular responses.
• Advances in technology allow for unprecedented insights into dynamic cellular regulations.

Conclusions:

• Multiplex live imaging is a powerful tool for dissecting complex cellular signaling.
• Continued progress in biosensor development, imaging, and computational analysis will enhance its utility in basic and translational research.
• This methodology is key to understanding cancer heterogeneity and therapeutic resistance.