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

描述（由申请人提供）：近红外光非常适合活细胞和整个动物的光学成像，因为在该光谱区域中自发荧光较低，光毒性较低，组织穿透性更强。然而，没有近红外荧光蛋白，并且外源近红外荧光团缺乏细胞渗透性或在细胞内产生广泛的背景标记。这极大地限制了使用近红外光对细胞内事件进行成像的能力。例如，示例性磺化近IR荧光团如Cy5不是细胞可渗透的，并且在很大程度上限于细胞外应用。此外，近红外荧光团已经取代了通常用于触发荧光激活的方法。这限制了近红外荧光团报告酶活性或响应光活化的能力。在这项资助中，我们计划一种合理而直接的方法：1）将示例性磺化近红外荧光团传递到细胞中，2）创建可用于研究生物分子和细胞动态行为的光活化近红外荧光团，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中的动态和酶促过程的研究带来近红外成像的显着优势，并具有基础研究和医学成像的主要潜在应用。公共卫生相关性：用光对细胞进行实时成像是观察细胞功能内部运作的一种简单而快速的方法。这项研究将提高光成像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.