Recent Advances in Fluorescent Probes for Imaging Enzyme Activity in NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA released by neutrophils during a specialized form of cell death called NETosis. Originally described as a mechanism of pathogen neutralization, NETs have since been implicated in a growing number of pathological conditions, including inflammatory diseases, autoimmunity, cancer, and neurodegeneration. The complexity and rapid dynamics of neutrophils and NET formation pose significant challenges for conventional approaches, highlighting a need for tools that can report molecular events with high spatial and temporal precision. Fluorescent probes have proven invaluable in this regard, offering both the sensitivity and selectivity required to study this process. This review provides a comprehensive overview of recent progress on the development of fluorescent probes for imaging NETosis, focusing on their design principles, biological applications, and key limitations. We discuss how these tools have advanced our understanding of the stimulus-dependent and spatiotemporally distinct activation dynamics of enzymes central to NETosis. We further highlight remaining challenges and outline design strategies that we believe will guide the development of next-generation tools to investigate neutrophil biology in health and disease.