Resonances at Fundamental and Harmonic Frequencies for Selective Imaging of Sine‐Wave Illuminated Reversibly Photoactivatable Labels

We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine‐wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non‐redundant resonant observables are simultaneously retrieved from a single experiment with phase‐sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance. Improving label discrimination: HIGHLIGHT (pHase‐sensItive imaGing of reversibly pHotoactivatable Labels after modulatIon of activatinG ligHT) is introduced as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with given kinetics. HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. It is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far.