Imaging of oxygen distributions is of key importance for several areas of physiology and medicine. For example, in neuroscience, the ability to image brain oxygenation is critical for understanding neuronal activation;[1
] in ophthalmology, imaging of retinal pO2
(partial oxygen pressure) can help elucidating causes of diabetic retinopathy and macular degeneration;[2
] in photodynamic therapy of cancer, measuring tumor pO2
will facilitate optimization of treatment protocols, complementing direct imaging of singlet oxygen.[3
] Oxygen levels can vary between individual tissue compartments (intravascular vs interstitial vs intracellular), and the ability to quantify changes in partial oxygen pressure (pO2
) between and within these compartments at the microscopic level would provide invaluable information for physiological research.
in biological systems can be measured optically by the phosphorescence quenching method,[4
] using probes with controllable quenching parameters and defined bio-distributions. Such probes are delivered directly into the medium of interest, where they serve as molecular sensors for oxygen. Phosphorescence quenching has been widely used to image oxygen in biological objects, including examples of microscopy.[6
] Wide area illumination and CCD-based detection are most commonly used in phosphorescence imaging, as they permit fast acquisition, although at the expense of spatial resolution and 3D capability. Confocal laser scanning microscopy has also been applied as an alternative method to image pO2
. However, either large pinholes (diameter ≈1 mm) were required to compensate for low rates of phosphorescence emission,[10
] resulting in low spatial resolution; or probes with short triplet lifetimes and, therefore, low oxygen sensitivity, had to be employed.[11
Herein we report a new approach to oxygen imaging, which combines principles of the phosphorescence quenching method with two-photon laser scanning microscopy (2P LSM).[12
] 2P excitation offers several advantages over linear methods, such as improved depth resolution for 3D imaging and reduced risk of photodamage.[13
] In principle, combining 2P LSM and phosphorescence quenching should be a straightforward task;[14
] however, phosphorescent probes, typically based on Pt and Pd porphyrins, posses extremely low two-photon absorption (2PA) cross-sections (σ2
] Using these probes, either exceedingly high excitation powers would be required to generate adequate signals, or thousands of phosphorescence decays would have to be averaged in each image pixel, resulting in unacceptably long acquisition periods.
In order to circumvent this problem, an approach to enhance triplet generation via 2P excitation in metalloporphyrins has been proposed,[15
] whereby excitation energy is captured by several 2P-antenna chromophores and transmitted to metalloporphyrin-core by Förster-type resonance energy transfer (FRET). Recent studies have shown that distances between the antenna and the core, as well as their redox potentials, must be carefully tuned in order to prevent intramolecular quenching of phosphorescence via electron transfer (ET).[17
Herein, we introduce the first practical 2P phosphorescent nanoprobe and demonstrate its application in oxygen imaging. The new technique is validated on microheterogeneously oxygenated phantoms with a priori known oxygen distributions and used to obtain pO2 images inside endothelial cells.