Live-cell intracellular immunofluorescence

摘要 免疫荧光技术是基础生物学研究和临床取样的重要手段之一 由于抗体的广泛可用性以及荧光成像的分辨率和灵敏度， 尽管IF在其最初发明以来的过去60年中产生了大量的数据，但IF遭受了 从一个基本的限制：不能询问活细胞中的细胞内目标（细胞必须是 首先固定，因此仅提供动态小区信令过程的快照）。考虑到3D结构， 在细胞中，具有生物学意义的细胞内靶点比它们的细胞膜对应物多得多， 但免疫试剂如抗体、抗体片段、肽、纳米抗体和scFv， 高度亲水的大分子，不能自发地穿过细胞膜。克服障碍 细胞膜和内吞作用，这是非常有效地防止完整的生物分子进入 细胞质，我们建议开发一种细胞内蛋白质递送技术，建立在一个独特的概念上： 共价胆固醇标记。与主要基于 内吞作用（由于捕获的成像剂产生的高背景， 在核内体中），我们的小分子标签使蛋白质能够渗透通过细胞膜。更 重要的是，这是在膜中不产生引起细胞毒性的孔的情况下实现的。如果成功，这 平台技术将为分子生物学、药物开发和细胞 工程.

ABSTRACT Immunofluorescence (IF) is one of the most important tools for both basic biology research and clinical sample imaging due to the broad availability of antibodies and the resolution and sensitivity of fluorescence imaging. Despite the tremendous amount of data produced by IF in the past ~60 years since its initial invention, IF suffers from a fundamental limitation: incapability of interrogating intracellular targets in live cells (cells have to be fixed first, thus only providing a snapshot of the dynamic cell signaling process). Considering the 3D structure of cells, there are far more intracellular targets with biological significance than their cell-membrane counterparts, but immunological agents such as antibodies, antibody fragments, peptides, nanobodies, and scFvs, being highly hydrophilic macromolecules, cannot spontaneously cross the cell membranes. To overcome the barriers of cell membrane and endocytosis that are extremely effective in preventing intact biomolecules to enter the cytosol, we propose to develop an intracellular protein delivery technology building on a unique concept: non- covalent cholesterol tagging. In contrast to conventional delivery technologies that are mostly based on endocytosis (inappropriate for live cell imaging due to the high background generated by imaging agents trapped in endosomes), our small-molecule tag enables proteins to permeate through the cell membrane. More importantly, this is achieved without generating pores in the membrane that cause cytotoxicity. If successful, this platform technology should open a whole new dimension for molecular biology, drug development, and cell engineering.