Department of Genetics, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, NY, 10461, USA.
Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.
Nat Commun. 2024 Sep 6;15(1):7788. doi: 10.1038/s41467-024-51857-x.
Near-infrared (NIR) probes are highly sought after as fluorescent tags for multicolor cellular and in vivo imaging. Here we develop small NIR fluorescent nanobodies, termed NIR-Fb and NIR-Fb, enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb, to simultaneously target several antigens within the NIR spectral range. Leveraging the antigen-stabilizing property of the developed NIR-Fbs, we then create two modular systems for precise control of gene expression in GFP-labeled cells. Applying the NIR-Fbs in vivo, we target cells expressing GFP and the calcium biosensor GCaMP6 in the somatosensory cortex of transgenic mice. Simultaneously tracking calcium activity and the reference signal from NIR-Fbs bound to GCaMP6 enables ratiometric deep-brain in vivo imaging. Altogether, NIR-Fbs present a promising approach for imaging and manipulating various processes in live cells and behaving animals expressing GFP-based probes.
近红外 (NIR) 探针作为多色细胞和体内成像的荧光标记物备受关注。在这里，我们开发了小型 NIR 荧光纳米抗体，分别命名为 NIR-Fb 和 NIR-Fb，可实现各种 GFP 衍生探针和生物传感器的无背景可视化。我们还设计了一个红移变体 NIR-Fb，可同时在近红外光谱范围内靶向几个抗原。利用开发的 NIR-Fbs 的抗原稳定特性，我们随后创建了两个用于精确控制 GFP 标记细胞中基因表达的模块化系统。在体内应用 NIR-Fbs 时，我们靶向在转基因小鼠感觉皮层中表达 GFP 和钙生物传感器 GCaMP6 的细胞。同时跟踪钙活性和与结合到 GCaMP6 的 NIR-Fbs 相关的参考信号，可实现比率式深部脑体内成像。总之，NIR-Fbs 为成像和操纵表达 GFP 探针的活细胞和行为动物中的各种过程提供了一种有前途的方法。
