DENDRITIC OXYGEN SENSOR WITH TWO-PHOTON ABSORBING ANTENNA

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 将磷光猝灭技术与多光子激光扫描显微镜相结合，可以实现亚微米空间分辨率的三维氧成像。 然而，传统上用作生物氧传感中的磷光体的基于Pt和Pd卟啉的磷光染料表现出极低的双光子吸收（2 PA）截面。 使用已知的大的双光子截面连接到卟啉的发色团，我们正在研究的可能性，产生高的磷光产量后，光激发和随后的能量转移发色团和卟啉之间。这样的复合物对于采用磷光而不是荧光的单分子（SM）研究是高度期望的。到目前为止，由于信号水平低，磷光的使用在SM技术中相当有限。 氧依赖磷光猝灭法是一种灵敏的测定氧分子的方法。我们提出的复合物可以发挥关键作用，在体内采用成像技术测量组织中的分子氧。 我们项目的具体目标包括： (1)合成2 P吸收磷光探针，其中2 P天线染料通过树枝状“绝缘体”与三重态发射体分离，以防止通过电子转移的磷光的分子内猝灭。 (2)修改现有的共聚焦显微镜系统，将其与飞秒钛：蓝宝石激光器和时域磷光计耦合，以执行天线发色团的2 P激发，在表面下的轴向平面中扫描物体并测量磷光寿命。 (3)使用具有显微氧气梯度的适当模型对齐并测试整个设置。 通过在大鼠背窗肿瘤模型中进行体内氧成像来验证该技术。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Imaging oxygen in 3D with sub-micron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. However, Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen sensing, exhibit extremely low two-photon absorption (2PA) cross-sections. Using chromophors with large known two photon cross sections linked to porphyrins, we are investigating the possibility of generating high phosphorescence yields after photoexcitation and subsequent energy transfer between chormophor and porphyrin. Such complexes would be highly desirable for single molecule (SM) studies employing phosphorescence instead of fluorescence. Up to this point, the use of phosphorescence is quite limited in SM techniques due to the low level of signal. Oxygen dependent quenching of phosphorescence is a sensitive method for measuring molecular oxygen. Our proposed complexes could play a key role in measuring molecular oxygen in tissue employing an imaging technique in vivo. Specific aims of our project include: (1) Synthesize 2P absorbing phosphorescent probes, in which 2P antenna dyes are separated from the triplet emitter by a dendritic "insulator," to prevent intamolecular quenching of the phosphorescence via electron transfer. (2) Modify the existing confocal microscope system by coupling it with a femtosecond Ti:Sapphire laser and a time domain phosphorometer to perform 2P excitation of antenna chromophores, scanning the object in axial planes under the surface and measuring phosphorescence lifetimes. (3) Align and test the entire setup using an appropriate model with microscopic oxygen gradients. Validate the technique by performing in vivo oxygen imaging in the dorsal window tumor model in rats.