Li Bing, Ponjavic Aleks, Chen Wei-Hsin, Hopkins Lee, Hughes Craig, Ye Yu, Bryant Clare, Klenerman David
Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge CB2 1EW, UK.
Department of Veterinary Medicine, University of Cambridge, Madingley Rd, Cambridge CB3 0ES, UK.
Anal Chem. 2021 Mar 2;93(8):4092-4099. doi: 10.1021/acs.analchem.0c05296. Epub 2021 Feb 17.
The detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, the delivery of single molecules remains to be a challenge. Currently, there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position and then image the cellular response. Here, we have combined light-sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local-delivery selective-plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and ionic feedback current at the nanopipette tip to control the position from which the molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2 μm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited the aggregates of amyloid-β, which causes proteotoxicity relevant to Alzheimer's disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in the three-dimensional (3D) mode enables the live detection of MyDD88 accumulation and the formation of myddosome signaling complexes, as a result of the aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.
在过去十年中，生物系统中单分子检测的分辨率迅速提高。然而，单分子的递送仍然是一个挑战。目前，尚无有效的方法能够在确定的位置将精确数量的分子引入单个细胞或细胞内，然后对细胞反应进行成像。在此，我们将光片显微镜与使用纳米移液器的局部递送相结合，以将单个蛋白质精确递送至确定的位置。我们将此方法称为局部递送选择性平面照明显微镜（ldSPIM）。ldSPIM使用纳米移液器和纳米移液器尖端的离子反馈电流来控制分子递送的位置。递送的蛋白质数量可通过改变施加的电压来控制。对于单分子检测，我们使用反射原子力显微镜悬臂实现了单物镜SPIM，以创建一个2μm厚的薄片。使用此设置，我们证明ldSPIM可以将单个荧光标记的蛋白质递送至HK293细胞的质膜上或细胞质中。接下来，我们将与阿尔茨海默病相关的蛋白毒性的淀粉样β聚集体沉积到稳定表达MyDD88-eGFP融合构建体的单个巨噬细胞上。三维（3D）模式下的全细胞成像能够实时检测MyDD88的积累以及髓样小体信号复合物的形成，这是聚集体诱导的Toll样受体4触发的结果。总体而言，我们展示了一种新型多功能成像系统，该系统能够将单个蛋白质精确递送至细胞表面或细胞质内的特定位置，并在活细胞内以单分子分辨率进行高速3D检测。
