Two-photon phosphorescence lifetime microscopy (2PLM) for high resolution imaging of oxygen

We present a new method for imaging of oxygen in biological samples with micron-scale resolution and three-dimensional capability. Our technique combines phosphorescence quenching with multiphoton laser scanning microscopy. It is termed two-photon phosphorescence lifetime microscopy (2PLM). 2PLM is made possible due to specially designed dendritic probes, in which phosphorescence of metalloporphyrins upon two-photon excitation is enhanced by intramolecular Frster-type energy transfer from two-photon antennae chromophores, located in the same probe molecule. Oxygen sensitivity of the probes is regulated by dendritic encapsulation of the core metalloporphyrin, while peripheral PEG groups on the dendrimer isolate the probes from contacts with biological macromolecules (e.g. proteins, nucleic acids etc) in the environment. Instrumentation and resolution of the method are discussed along with approaches to intracellular delivery of the probes. 2PLM was validated in pilot imaging experiments and used to obtain depth-resolved high-resolution oxygen maps in vivo in the brain.