DENDRITIC OXYGEN SENSOR WITH TWO-PHOTON ABSORBING ANTENNA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 将磷光猝灭技术与多光子激光扫描显微镜相结合，可以实现亚微米级空间分辨率的三维氧成像。然而，传统上用于生物氧传感的铂、钯基磷光染料表现出极低的双光子吸收(2PA)截面。 利用已知的与卟啉相连的大双光子截面的发色团，我们正在研究在光激发和随后的发色团和卟啉之间的能量转移后产生高磷光产量的可能性。这种络合物对于使用磷光而不是荧光的单分子(SM)研究是非常可取的。到目前为止，由于信号电平低，磷光在SM技术中的使用相当有限。 磷光依赖氧猝灭是一种灵敏的测量分子氧的方法。我们提出的络合物可以在利用体内成像技术测量组织中的分子氧方面发挥关键作用。 我们项目的具体目标包括： (1)合成2P吸收磷光探针，其中2P天线染料通过树枝状“绝缘体”从三重态发射体中分离出来，以防止通过电子转移对磷光进行分子内猝灭。 (2)对现有的共焦显微镜系统进行了改进，将其与飞秒钛宝石激光器和时间域磷光仪耦合，实现了对天线发色团的2P激发，扫描了物体表面下的轴向平面，并测量了磷光寿命。 (3)使用具有微观氧气梯度的适当模型对整个设置进行对准和测试。通过在大鼠背窗肿瘤模型中进行活体氧气成像来验证该技术。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.