Visualizing liquid-liquid phase separation and protein aggregates

Liquid-liquid phase separation (LLPS) facilitates functionally-relevant proteins to gather in high concentrations at specific locations. Several interacting proteins encapsulated in the membraneless droplets tend to form abnormal protein aggregates which are linked to numerous pathological diseases. Thus direct observation of LLPS, successive protein aggregates and the potential conversion processes is significant for understanding associated cell function and pathology. In this review, we firstly introduce the techniques for imaging LLPS, including in vitro reconstitution and intracellular visualizing strategies. Then we elaborate recent advances in the development of the imaging approaches for protein aggregates, followed by highlighting the monitor of protein conformational changes during aggregation process. Furthermore, the similarities and differences are discussed along with these state-of-the-art technologies. Finally, the major challenges and potential in exploration of detection methods for visualizing LLPS and protein aggregates are also pointed out. With these advances, it is becoming possible both to ‘see’ independent state of LLPS or protein aggregates, and realize the real-time monitor of the whole process. Direct observation of liquid–liquid phase separation (LLPS) and resulting protein aggregation plays a crucial role in understanding the associated mechanisms and progression. Here, the authors review advanced imaging techniques for visualizing LLPS and protein aggregates, offering insights into real-time monitoring of phase-separated processes.