No-wash live cell imaging with a photoswitchable near-infrared hCRBPII protein-fluorophore tag.

Fluorescence cell imaging provides a powerful tool to study biological processes including regulation, protein-protein interaction, trafficking, development, cellular structure and morphology, to name a few. Complimentary to fluorescent proteins (FPs), the development of multiple site-selective labeling techniques offer choice and flexibility in selection of fluorophores for optimal experimental design. Near-infrared (NIR) labels are highly desired since they enable deeper imaging depths and cleaner optical windows. Photochromic labels are also desirable since they provide the capability to control the fluorescence "turn-ON" and in some cases "turn-OFF" functionality. In addition, no-wash labeling techniques can greatly simplify experimental procedures and offer real-time imaging options. Also, compared to most of the regular FPs, these systems are often matured rapidly and do not need molecular oxygen for activation. Here, we present a no-wash photochromic NIR fluorescence live cell imaging approach. This method uses engineered human Cellular Retinol Binding Protein II (hCRBPII) as a genetically encodable tag and a solvatochromic dye FR-1V as the fluorophore. At the heart of this system, a photo-triggered switching between NIR "OFF" and "ON" modes provide spatiotemporal control for subcellular fluorescence imaging.