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 ~88 h to synthesize the probes, ~24 h to characterize the probes in solution and ~25 h to carry out the biological fluorescence imaging experiments in cells and liver tissue slices.

甲醛（FA）是可以在体内自发产生的最简单的活性羰基物质，在人类的认知能力和空间记忆中起着重要的作用。但是，过量摄入FA可能会导致一系列疾病，包括癌症，糖尿病，心脏和肝脏疾病以及各种神经病。因此，探索灵敏且快速的FA诊断方法对于了解和诊断这些疾病至关重要。近来，由于荧光探针的高选择性，快速响应，方便的操作和相对无创的性质，其已被越来越多地用作检测广泛范围的不同小分子的有力工具。因此，我们开发了两种基于萘二甲酰亚胺的荧光探针，用于检测细胞和溶酶体中的FA。与其他FA荧光探针相比，这两种探针具有几个优点，包括高灵敏度和选择性，出色的双光子特性以及高信噪比。在该协议中，我们提供了两种探针合成的详细步骤；表征其敏感性，选择性和溶液稳定性；和检测活细胞和小鼠肝组织切片中FA的代表性应用程序。该协议需要约88 h来合成探针，需要约24 h来表征溶液中的​​探针，并需要约25 h才能在细胞和肝组织切片中进行生物荧光成像实验。表征其敏感性，选择性和溶液稳定性；和检测活细胞和小鼠肝组织切片中FA的代表性应用程序。该协议需要约88 h来合成探针，需要约24 h来表征溶液中的​​探针，并需要约25 h才能在细胞和肝组织切片中进行生物荧光成像实验。表征其敏感性，选择性和溶液稳定性；和检测活细胞和小鼠肝组织切片中FA的代表性应用程序。该协议需要约88 h来合成探针，需要约24 h来表征溶液中的​​探针，并需要约25 h才能在细胞和肝组织切片中进行生物荧光成像实验。