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

近红外光非常适合于活细胞和整体的光学成像 动物因为较低的自发荧光，较低的光毒性和较大的组织 在这个光谱区域的穿透。然而，没有近红外荧光蛋白， 而外源近红外荧光团要么缺乏细胞通透性，要么导致 单元格内广泛的背景标记。这极大地限制了使用 近红外光成像细胞内事件。例如，示例性磺化近红外 像Cy5这样的荧光团不是细胞渗透性的，并且在很大程度上被限制在 细胞外应用。此外，近红外荧光团一直顽固地 通常用来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对酶反应的能力 光激活。在这笔赠款中，我们计划采取合理而直接的方法来1) 将典型的磺化近红外荧光团送入细胞，2)产生可光激活的 可用于研究生物分子动力学行为的近红外荧光团 和细胞，以及3)使用近红外光检测活细胞中的酶活性。团结在一起， 我们开发的近红外荧光工具将带来近红外的显著优势 成像研究活细胞的动态和酶过程，主要 在基础研究和医学成像方面的潜在应用。

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.