2025/01/17 by Hélène Merceron, Ian Coghill, Aliénor Lahlou +3 · 1 voice · 1 citation
Biochemistry, Genetics and Molecular Biology · Chemistry · Neuroscience · #Advanced Fluorescence Microscopy Techniques #Photoreceptor and optogenetics research #Spectroscopy and Laser Applications
paper · pdf · doi:10.1002/advs.202413291
openalex publication_date 2025/01/17 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/31
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.