Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.
Keywords: cell-to-cell variability; cyclic immunofluorescence; fluorescent proteins; high-throughput microscopy; immunofluorescence; live cell imaging; multicolor imaging; multispectral imaging; spatial organization; spectral unmixing.
© 2016 International Society for Advancement of Cytometry.