Enzyme regulation is crucial in living organisms to catalyze various biosyntheses to maintain several physiological functions. On the contrary, abnormal enzyme activities can affect bioactivities leading to various serious disorders including cancer, Alzheimer's disease, Parkinson's disease, heart disease, and so on. This biological significance led to the development of various techniques to map specific enzyme activities in living systems to understand their role and distribution. Two-photon microscopy (TPM) in particular has emerged as a promising system for in situ real-time bioimaging owing to its robustness, high sensitivity, and noninvasiveness. It was achieved through the use of a two-photon (TP) light source of an optical window (700-1450 nm) beneficial in deeper light penetration and extraordinary spatial selectivity. Therefore, developing enzyme sensors utilized in TPM has significance in obtaining in vivo enzyme activities with minimal perturbation. The development of an efficient detection tool for enzymes has been continuously reported in the previous literature; here, we meticulously review the TP design strategies that have been attempted by researchers to develop enzyme TP fluorescent sensors that are proving very useful in providing insights for enzyme investigation in the biological system. In this review, the representative TP enzymatic probes that have been made in the past 5 years and their applications in tissue imaging are discussed in brief. In addition, the prospects and challenges of TP enzymatic probe development are also discussed.
Keywords: biological imaging; enzyme detection; fluorescence; fluorophores; probes; rodent models; tissue imaging; two-photon microscopy.