Oxygen microscopy by two-photon excited phosphorescence

描述(由申请人提供)：体内氧气成像在现代生理学和医学中是一个具有挑战性的重要问题。过去已经提出了许多方法来绘制生物对象中的氧合关系图，但没有一种方法提供具有三维能力的显微成像手段。我们建议发展一种将磷光猝灭方法与双光子激光扫描显微镜(2P LSM)相结合的生物系统中氧的显微成像技术。工作计划有三个主要目标：1)双光子可激发磷光探测器的合成开发及其光物理表征；2)建造磷光双光子成像系统(磷光探头的长辐射寿命使双光子成像标准系统不适合这一应用)；3)使用关于氧在微观(亚细胞)一级的分布的信息具有高度意义的生物系统来测试这项技术。这些系统是：吞噬过程中形成的氧气梯度(S.Muro，宾夕法尼亚大学药理学)；眼睛脉络膜和视网膜之间的氧气梯度(E.Pugh，宾夕法尼亚大学眼科)；大脑中的氧气分布(D.Boas，哈佛/麻省理工学院)。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)是由美国国立卫生研究院资助的试点项目R21 EB-003663-01的延续。该传感器的设计结合了目前在几个纳米技术应用中受到仔细审查的原理和方法：由多个发色团组成的天线阵列的双光子吸收(2PA)，耦合到同一功能核心；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。验证了该设计的可行性，并确定了用于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改变，以允许对磷光进行成像。选择生物系统来评估技术的性能，同时为相关的生物医学问题获得有价值的信息。

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.