Lifetime imaging of singlet oxygen NIR phosphorescence

1O2 is the first excited state of molecular oxygen and the key intermediate in photosensitized oxidation reactions. Although 1O2 emits near-infrared (NIR) phosphorescence, direct microscopic observation of its emission is severely hampered by its low emission quantum yield, the competitive kinetics between its decay and diffusion, and the limited efficiency of NIR detectors. Here, we describe a 1O2 phosphorescence lifetime imaging microscope (1O2-PLIM) that allows acquiring lifetime and intensity profiles of 1O2 phosphorescence emission with improved resolution. Calibration carried out with photosensitizer solutions returned the expected lifetimes for 1O2 generation and decay. Nanometer-sized beads allowed the reconstruction of the excitation volume and the estimation of detection limit as 1 million 1O2 molecules in 60 fL of ethanol. Scanning samples in a confocal configuration provided intensity and lifetime image reconstruction of 1O2 emission from complex systems such as micrometer-sized polymer beads bound to photosensitizers, as well as from HaCaT keratynocytes previously incubated with a photosensitizer. Raw image data was corrected for the 1O2 emission lifetime and diffusion pathway within the confocal volume, using a mathematical model specifically developed for this purpose. This advancement in 1O2 imaging enables a better understanding and control of light-mediated reactions across chemistry, biomedicine, and environmental science. Obtaining precise values of 1O2 lifetimes in complex environments has remained a challenge due to a low emission quantum yield and competitive decay and diffusion kinetics. Here, the authors report a 1O2 phosphorescence lifetime imaging microscope (1O2-PLIM) that facilitates the acquisition of lifetime and intensity profiles of 1O2 phosphorescence emission with improved resolution, demonstrating its application to the study of micrometer-sized polymer beads bound to photosensitizers and HaCaT keratynocytes previously incubated with a photosensitizer.