Leadfluor-1 (LF1) is a small-molecule fluorescent sensor for detecting lead in biological and environmental samples, including live cells. This dye uses a xanthenone fluorescent scaffold coupled to a dicarboxylate pseudocrown ether receptor to achieve selective detection of Pb2+ in the presence of biologically relevant metal ions, including divalent calcium, magnesium and zinc. LF1 fluorescence increases by up to 18-fold on binding Pb2+. In this protocol, we describe the synthesis and application of LF1 to imaging lead accumulation within live cells. The preparation of LF1 is anticipated to take 14–21 d, and the imaging assays can be performed in 1–2 d with cultured cells.