Lifetime imaging of singlet oxygen NIR phosphorescence

¹ O ₂ is the first excited state of molecular oxygen and the key intermediate in photosensitized oxidation reactions. It is sensed by its near-infrared (NIR) phosphorescence emission that peaks at 1270 nm. However, several intrinsic properties of the ¹ O ₂ emission, such as the very low yield of photon emission, the comparable rates for decay and di!usion in dense media, and the low e”ciency of the cameras available in this wavelength range, make the acquisition of microscopic images of ¹ O ₂ localization a real challenge. Here, we describe a ¹ O ₂ phosphorescence lifetime imaging microscope ( ¹ O ₂ -PLIM) that allows acquiring lifetime and intensity profiles of ¹ O ₂ phosphorescence emission with micrometer resolution. Calibration carried out with photosensitizer solutions returned the expected lifetimes for ¹ O ₂ generation and decay. Nanometer-sized beads allowed the reconstruction of the excitation volume and the consequent estimation of detection limit as being 1 million ¹ O ₂ molecules in 2 femtoliters of ethanol. Scanning samples in a confocal configuration provided reconstruction of intensity and lifetime images of ¹ O ₂ 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 ¹ O ₂ emission lifetime and for the di!usion pathway within the confocal volume, using a mathematical model specifically developed for this purpose. This work provides a proof of principle for ¹ O ₂ imaging that will stimulate development of novel instrumentation, allowing a better understanding of how light interacts with matter and a!ects processes relevant to biology, medicine, agriculture, food and materials science.