Oxygen microscopy by two-photon excited phosphorescence

描述（由申请人提供）：体内氧成像是现代生理学和医学中具有挑战性的重要问题。过去已经提出了许多用于绘制生物物体中氧合的方法，但没有一种提供具有三维能力的显微成像方法。我们建议开发一种技术，在生物系统中的氧气显微成像耦合的磷光猝灭方法与双光子激光扫描显微镜（2 P LSM）。本工作计划有三个主要目标：1）双光子激发磷光探针的合成及其物理表征; 2）磷光双光子成像系统的构建（磷光探针的长辐射寿命使得用于双光子成像的标准系统不适合于该应用）; 3）使用生物系统测试该技术，对于该生物系统，关于微观（亚细胞）水平的氧分布的信息具有高度重要性。这些系统是：在吞噬细胞内吞作用期间形成的氧梯度（S。Muro，Penn Pharmacology）;眼脉络膜和视网膜之间的氧梯度（E. Pugh，Penn Ophthalmology）;脑中的氧分布（D. Boas，哈佛/MGH）。双光子磷光氧纳米传感器的开发是该提案的关键，工作计划的相应部分（具体目标1和2）是由NIH拨款R21 EB-003663-01资助的试点项目的延续。传感器的设计结合了目前在几个纳米技术应用中受到审查的原理和方法：由天线阵列的双光子吸收（2 PA），由多个发色团组成，耦合到同一个功能核心;从天线到核心的分子内能量转移（ET）;核心的树枝状封装，以实现其保护和控制其局部环境。该设计的可行性已被证明，并已确定在体内应用的探针的优化方法。此外，所获得的结果揭示了必须对双光子标准成像设置进行的更改，以允许磷光成像。生物系统的选择，以评估该技术的性能，并同时获得相关的生物医学问题的有价值的信息。

DESCRIPTION (provided by applicant): Imaging oxygen in vivo presents a challenging and important problem in modern physiology and medicine. A number of methods for mapping oxygenation in biological objects have been proposed in the past, but none of them provides means for microscopic imaging with three-dimensional capability. We propose to develop a technique for microscopic imaging of oxygen in biological systems by coupling the phosphorescence quenching method with two-photon laser scanning microscopy (2P LSM). The work plan has three main objectives: 1) synthetic development of two-photon excitable phosphorescent probes and their photophysical characterization; 2) construction of two-photon imaging system for phosphorescence (long radiative lifetimes of phosphorescent probes make standard systems for two-photon imaging non- suitable for this application); 3) testing the technique using biological systems for which information about the distribution of oxygen at microscopic (sub-cellular) level is of high significance. These systems are: oxygen gradients formed-during phagocytic endocytosis (S. Muro, Penn Pharmacology); oxygen gradient between the choroid and the retina of the eye (E. Pugh, Penn Ophthalmology); oxygen distribution in the brain (D. Boas, Harvard/MGH). Development of two-photon phosphorescent oxygen nanosensors is the key to the proposal, and the corresponding part of the work plan (Specific Aims 1 and 2) is a continuation of the pilot project funded by the NIH grant R21 EB-003663-01. The design of the sensor combines principles and approaches currently under scrutiny in several nanotechnology applications: two-photon absorption (2PA) by an antenna-array, consisting of multiple chromophores, coupled to the same functional core; intramolecular energy transfer (ET) from the antenna to the core; dendritic encapsulation of the core to achieve its protection and to control its local environment. The feasibility of the design has been demonstrated and the ways of optimization of probes for in vivo applications have been identified. Furthermore, the results obtained reveal changes that will have to be made to two-photon standard imaging setup to permit imaging of phosphorescence. The biological systems are selected to evaluate the performance of the technique and to simultaneously obtain valuable information for the related bio-medical problems.