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Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane