Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany.
Center for Biostructural Imaging of Neurodegeneration (BIN), University of Göttingen Medical Center, Göttingen, Germany.
Nat Commun. 2024 Oct 10;15(1):8771. doi: 10.1038/s41467-024-53030-w.
Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, which have been revealed using primary antibodies and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional antibodies revealed by engineered secondary nanobodies that allow the selective removal of fluorescence signals. We develop three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5-8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.
荧光显微镜在生物科学领域一直是一种具有变革性的技术。然而，大多数应用都局限于少数已通过初级抗体和荧光偶联的二级抗体揭示的靶点。超分辨率技术，如交换-PAINT 和最近的 SUM-PAINT，提高了多重检测能力，但它们需要专用设备、软件和知识。为了在任何实验室的任何成像技术中实现多重检测，我们开发了 NanoPlex，这是一种基于工程化的二级纳米抗体揭示的常规抗体的简化方法，该方法允许选择性去除荧光信号。我们开发了三种互补的信号去除策略：OptoPlex（光诱导）、EnzyPlex（酶）和 ChemiPlex（化学）。我们展示了 NanoPlex 可以用于 3D 共聚焦分析的 21 个靶点，以及用于 dSTORM 和 STED 超分辨率成像的 5-8 个靶点。NanoPlex 有可能彻底改变多靶标荧光成像方法，可能重新定义基于抗体的分析的多重检测能力。
