Periodic Light Modulations for Low-Cost Wide-Field Imaging of Luminescence Kinetics Under Ambient Light

Imaging luminescence kinetics is invaluable in many fields, including biology and chemistry. However, the luminescence lifetime of most photo-activated states is in the low ns-µs range and its measurement requires adding costly image intensifiers to cameras to access the fast phenomena present. Here, the Rectified Imaging under Optical Modulation (RIOM) and Heterodyne Imaging under Optical Modulation (HIOM) protocols make this possible with standard low-cost cameras only, even under ambient light. RIOM and HIOM originate from a thorough theoretical analysis, which showed that modulated illumination of any reversibly photoactivable luminophore probed at high frequency generates components of the luminescence response that are detectable at low frequency: RIOM harnesses the mean luminescence response to modulated light at a single frequency, whereas HIOM exploits the luminescence response to modulated light at two close frequencies. When the luminophore behavior obeys a two-state model, the frequency dependence of the RIOM and HIOM observables can be used to extract a photoactivation time. In this work, their ability to retrieve maps of the luminescence lifetime of a reversibly photoswitchable protein, and phosphorescent microsensors are demonstrated. Where the luminophore does not obey a two-state model, RIOM and HIOM can still retrieve rich information, as here exemplified by the recovery of kinetic fingerprints of the physiological state of a plant.