Cellular senescence is a state of stable cell cycle arrest in response to various cellular stresses that are involved in aging and age-related diseases. However, detection of senescence remains largely in vitro or ex vivo due to limited tools for sensitive detection of senescence in vivo. Two-photon microscopy has the advantages of deeper tissue penetration, excellent spatial resolution and efficient light detection with reduced photobleaching effects. Herein, we describe the development of a merocyanine-based two-photon fluorogenic probe that can be activated by senescence-associated β-galactosidase (SA-β-gal), the most widely used senescence marker, for the detection of cellular senescence. Upon activation, the probe exhibited a remarkable fluorescence enhancement via modulating the internal charge transfer (ICT) effect. Furthermore, the specificity was evaluated, and the results indicated that it displays an excellent selectivity for β-galactosidase over other enzymes and biologically related molecules. Subsequently, we tested our probe to visualize β-galactosidase activity in the CT26.CL25 cell line, an engineered cell line that stably expresses β-galactosidase; compared with the control wide type cell line, a dramatic fluorescence increase was observed. The probe’s efficiency in differentiating senescent cells induced by chemotherapeutic drugs and healthy cells is evaluated in cell and animal models.

Activatable two-photon fluorogenic probe for real-time imaging of cellular senescence

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