Dendritic upconverting nanoparticles for multiphoton imaging and sensing

描述（由申请人提供）：在这个项目中，我们解决了双光子显微镜对探针的需求，双光子显微镜是一种领先的成像技术，用于在微米尺度的空间分辨率下动态可视化和定量体内生物过程的三维成像。我们建议开发一类新的多光子探针，称为树突状UCNPs，它包括镧系上转换纳米粒子（UCNPs）和树突状配体。UCNPs的关键优势在于其极高的多光子吸收截面，比目前可用的最有效的多光子探针高出几个数量级。最近，我们证明了由于这种显著的特性，可以使用简单的低功率连续波（CW）红外光源完成体内双光子深度分辨显微成像，这与需要非常昂贵的脉冲飞秒激光器的传统双光子实验形成鲜明对比。这一显著的优势来自于UCNPs的其他优点，包括创纪录的高光稳定性，零背景荧光（由于连续红外激发）和大大降低光损伤的风险。然而，缺乏强大的UCNP溶解和功能化方法一直是阻碍其纳入现代成像方法工具包的主要障碍。我们建议用树突大分子来解决这个问题。我们的关键主张是，用亲水的形状持久的树状大分子修饰UCNP表面，将构成一种高效和通用的可溶生物相容性UCNP的途径，其发光将通过UCNP到树状大分子的激发能转移（EET）与分析物检测相耦合。我们的方法利用了树突结构的独特结构特征，即内在多价和伪球形。将开发用于形态血管造影双光子成像的“无色”探针和用于特定分析物（pH和Ca2+）成像的专用探针。为了测试探针，我们将进行：(a)啮齿动物大脑的活体血管造影成像，确定功能刺激下血液流变学的变化；(b)脑卒中啮齿动物模型中组织pH和氧分压（pO2）变化的同步体内多光子成像；d)电刺激小鼠脑垂体细胞外Ca2+通量成像。所有这些实验将不同于传统的多光子成像，激发源的成本将降低约1000倍。这些应用将证明新探针的能力：a)取代目前使用的昂贵的多光子装置；b)超越并解决神经科学中目前没有替代解决方案的问题和假设。

DESCRIPTION (provided by applicant): In this project we address the need in probes for two-photon microscopy - the leading imaging technique for dynamic visualization and quantification of biological processes in vivo in 3D with micron-scale spatial resolution. We propose to develop a new class of multiphoton probes, termed dendritic UCNPs, which comprise lanthanide-based up converting nanoparticles (UCNPs) and dendritic ligands. The key advantage of UCNPs is their enormously high multiphoton absorption cross-sections, which exceed those of the most efficient multiphoton probes available today by several orders of magnitude. Recently, we demonstrated that due to this remarkable property, in vivo two-photon depth-resolved microscopic imaging with UCNPs can be accomplished using simple low-power continuous-wave (CW) infrared light sources, which is in contrast to conventional two-photon experiments requiring very expensive pulsed femtosecond lasers. This remarkable advantage comes on top of other benefits of UCNPs, which include record-high photo stability, zero background fluorescence (due to CW infrared excitation) and greatly diminished risk of photo damage. However, lack of robust methods of UCNP solubilization and functionalization has been a major obstacle preventing their inclusion into the toolkit of modern imaging methods. We propose to solve this problem by using dendritic macromolecules. Our key proposition is that modification of UCNP surfaces with hydrophilic shape-persistent dendrimers will make up an efficient and general route to soluble bio-compatible UCNPs, whose luminescence will be coupled to analyte detection via UCNP-to-dendrimer excitation energy transfer (EET). Our approach capitalizes on unique structural features of dendritic architecture, i.e. intrinsic polyvalency and pseudo- globular shape. Both "colorless" probes for morphologic angiographic two-photon imaging and dedicated probes for imaging of specific analytes (pH and Ca2+) will be developed. To test the probes we will perform: (a) angiographic imaging in vivo in rodent brain, determining changes in blood rheology upon functional stimulation; (b) simultaneous in vivo multiphoton imaging of alterations in tissue pH and partial pressure of oxygen (pO2) in stroke rodent models; d) imaging of extracellular Ca2+ flux in mouse neurohypophysis upon electrical stimulation. All these experiments will differ from conventional multiphoton imaging in that the cost of the excitation sources will be lower by ca 1000 fold. These applications will demonstrate the ability of the new probes to a) replace currently used expensive multiphoton setups; and b) go beyond and address questions and hypotheses in neuroscience for which no alternative solutions are currently available.