Recent advances in fluorescent probes development for interrogating protein misfolding and aggregation

Protein folding integrity in live cells is maintained by the protein homeostasis network, which includes molecular chaperones and degradation pathways. Failures in protein folding are implicated in a variety of human disorders, often resulting in protein aggregation and loss of function. While studies using purified proteins ex vivo provide valuable insights, the behavior of these proteins in living cells is significantly more complex than in simplified models. Therefore, advanced biomedical imaging techniques are essential for real-time, dynamic, and in situ monitoring of these phenomena in the cellular environment, particularly through fluorescence-based methods. This review discusses the use of small-molecule fluorescent probes to detect protein misfolding and aggregation in both in vitro and in vivo settings. Additionally, it highlights emerging design strategies for novel probes based on excited-state behavior. The ongoing development of these tools is expected to enhance our understanding of protein homeostasis and stress. Ultimately, advances in this field may contribute to the elucidation of disease mechanisms and facilitate the discovery of new therapeutic strategies.