1. Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation.
- Author
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Barykina, Natalia V., Carey, Erin M., Oliinyk, Olena S., Nimmerjahn, Axel, and Verkhusha, Vladislav V.
- Subjects
CELL imaging ,IMMUNOGLOBULINS ,TRANSGENIC mice ,BIOSENSORS ,GENE expression - Abstract
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
LAG16 and NIR-FbLAG30 , enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb(718) , 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-FbLAG s bound to GCaMP6 enables ratiometric deep-brain in vivo imaging. Altogether, NIR-FbLAG s present a promising approach for imaging and manipulating various processes in live cells and behaving animals expressing GFP-based probes. Near-infrared (NIR) probes are in high demand as fluorescent tags for multicolour cellular and in vivo imaging. Here authors develop series of NIR nanobodies that bind to GFP-based biosensors, and demonstrate ratiometric deep-brain in vivo imaging of active GCaMP6f-expressing neurons. [ABSTRACT FROM AUTHOR]- Published
- 2024
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