Applications of fluorescence lifetime imaging microscopy in brain disease research: A comprehensive review

Neurodegenerative diseases (NDs) and brain tumors constitute two of the most devastating classes of brain disorders, imposing severe and growing burdens on global public health. Fluorescence lifetime imaging microscopy (FLIM) represents a powerful, environment-sensitive analytical tool that enables quantitative interrogation of molecular excited-state decay dynamics, independent of fluorophore concentration. This technique offers unprecedented insights into diverse biological processes, including metabolic reprogramming, protein–protein interactions, and intracellular microenvironmental dynamics, thereby providing a unique mechanistic window into the pathogenesis and early diagnosis of brain diseases. This review provides a comprehensive synthesis of state-of-the-art applications of FLIM in neuropathological research. We first introduce the fundamental principles and major detection modalities of FLIM. We then highlight key advances in applying FLIM to the study of representative brain diseases, with a focus on probing pathological protein aggregation in NDs and delineating tumor margins in brain malignancies. Finally, we critically evaluate current limitations and outline promising future directions to advance FLIM-based technologies toward clinical translation in brain health care.