Preparation of robust fluorescent probes for tracking endogenous formaldehyde in living cells and mouse tissue slices

Formaldehyde (FA) is the simplest active carbonyl species that can be spontaneously produced in the body and plays important roles in human cognitive ability and spatial memory. However, excessive intake of FA may cause a series of diseases, including cancer, diabetes, heart and liver diseases and various neuropathies. Hence, the exploration of sensitive and fast detection methods for FA is crucial to understand and diagnose these diseases. Recently, fluorescent probes have been increasingly employed as powerful tools for detecting a broad range of different small molecules due to their high selectivity, rapid response, convenient operation and relatively non-invasive nature. Thus, we have developed two naphthalimide-based fluorescent probes for detecting FA in cells and in lysosomes. Compared with other FA fluorescent probes, these two probes have several advantages, including high sensitivity and selectivity, excellent two-photon properties and high signal-to-noise ratio. In this protocol, we provide detailed procedures for the synthesis of the two probes; characterization of their sensitivity, selectivity and stability in solution; and representative application procedures for detecting FA in living cells and mouse liver tissue slices. The protocol requires similar to 88 h to synthesize the probes, similar to 24 h to characterize the probes in solution and similar to 25 h to carry out the biological fluorescence imaging experiments in cells and liver tissue slices.