Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy

•A bacterial pepN activatable fluorescent biosensor based on ratiometric measurement.•The biosensor can be a tool to determine virulent bacteria in a few minutes.•NIR N-aminopeptidase fluorescent sensor useful in STED and excitable by two photons.•STED measurements show the hot spots where the enzymatic reaction begins.•We locate the sites where alanine amino peptidase is placed in bacteria.