Delivery and Triggered Activation of Near-IR Fluorophores for Live Cell Imaging

描述（由申请人提供）：近红外光非常适合于活细胞和整个动物的光学成像，因为在该光谱区域具有较低的自身荧光，较低的光毒性和较大的组织穿透性。然而，没有近红外荧光蛋白，外源性近红外荧光团要么缺乏细胞通透性，要么在细胞内引起广泛的背景标记。这极大地限制了使用近红外光成像细胞内事件的能力。例如，典型的磺化近红外荧光团，如Cy5，不具有细胞渗透性，主要局限于细胞外应用。此外，近红外荧光团对通常用于触发荧光激活的方法具有抗性。这限制了近红外荧光团报告酶活性或对光激活反应的能力。在这项资助中，我们计划一个合理和直接的方法，1)将典型的磺化近红外荧光团传递到细胞中，2)创建可光激活的近红外荧光团，可用于研究生物分子和细胞的动态行为，3)使用近红外光检测活细胞中的酶活性。总之，我们开发的近红外荧光工具将为活细胞动态和酶促过程的研究带来近红外成像的显着优势，在基础研究和医学成像方面具有重要的潜在应用。与公共卫生相关：用光对细胞进行实时成像是一种简单而快速的观察细胞功能内部运作的方法。本研究将提高光成像技术用于疾病研究和诊断的能力。

DESCRIPTION (provided by applicant): Near-IR light is ideally suited to the optical imaging of live cells and whole animals because of the lower autofluorescence, lower phototoxicity, and greater tissue penetration in this spectral region. However, there are no near-IR fluorescent proteins, and exogenous near-IR fluorophores either lack cell permeability or give rise to extensive background labeling within cells. This has greatly limited the ability to use near-IR light to image intracellular events. For example, exemplary sulfonated near-IR fluorophores such as Cy5 are not cell permeable and have been largely restricted to extracellular applications. Furthermore, near-IR fluorophores have been recalcitrant to the methods generally used to trigger the activation of fluorescence. This has limited the ability of near-IR fluorophores to report on enzymatic activity or be responsive to photoactivation. In this grant, we plan a rational and straightforward approach to 1) deliver exemplary sulfonated near-IR fluorophores into cells, 2) create photoactivatable near-IR fluorophores that can be used to study the dynamic behavior of biomolecules and cells, and 3) detect enzymatic activity in live cells using near-IR light. Together, the near-IR fluorescent tools we develop will bring the significant advantages of near-IR imaging to the study of dynamic and enzymatic processes in live cells, with major potential applications for both basic research and medical imaging. PUBLIC HEALTH RELEVANCE: Live imaging of cells with light is a simple and rapid way to observe the inner workings of cell function. This study will improve the ability of light-based imaging to be used for the study and diagnosis of disease.