An NIR emitting styryl dye with large Stokes shift to enable co-staining study on zebrafish neuromast hair cells

Abstract

Hearing loss is a significant public health problem, and the "loss of sensory hair cells" is one of two leading causes in humans. Advanced imaging reagents are desirable for understanding the role of the surrounding support cells in the loss or regeneration of the hair cells. A styryl dye was found to exhibit NIR emission (λ_em ≈ 684 nm) with a very large Stokes shift (Δν ≈ 9190 cm^-1), due to the incorporation of excited state intramolecular proton transfer (ESIPT) mechanism. When used to stain live zebrafish embryos, the probe was found to exhibit good selectivity in targeting neuromasts, which are sensory organs on the surface of the fish's body. The finding was verified by direct comparison with the known neuromast-labeling reagent, 4-Di-2-ASP. In contrast to the existing styryl dyes that label neuromast hair cells, the new probe labeled both neuromast hair cells and the surrounding support cells, while giving discernable signals. The study thus illustrated a useful tool to aid the developmental study of two closely related cell types on the mechanosensory sensory organ of zebrafish, which is a powerful animal model for hearing loss research.

Keywords: Cyanine; Excited state intramolecular proton transfer; Near Infrared (NIR) emission; Neuromasts; Styryl dye; Support cell; Zebrafish.

Figure 1.

(a) Sketch of the lateral line sensory organ on a zebrafish at 72 hpf. (b) The structure of an enlarged neuromast.

Figure 2.

Normalized UV-vis absorption (broken lines) and fluorescence (solid lines) spectra of 4-Di-2-ASP (a) and 1 (b) in CH₂Cl₂ and H₂O (10 μM solution) at 20°C. Excitation for 1 at 390 and 420 nm.

Figure 3.

UV-vis absorption λ_abs (a) and fluorescence λ_em (b) of 1, 2 and 4-Di-2-ASP in different solvents. All data were collected at 20°C.

Figure 4.

Confocal images of zebrafish embryos (40–72 hpf) labeled with 4-Di-2-ASP or probe 1. Anterior to the left and dorsal up for all images. Excitation wavelength was at 488 nm with a 595 nm filter (Texas Red) for 4-Di-2-ASP, and at 405 nm with filters at 700 nm (Texas Red and Cy5) for probe 1, respectively. Neuromasts and non-neuromasts labeling was indicated by arrowheads and arrows, respectively.

Fig. 5.

Confocal images of neuromasts from a live zebrafish, 3, 5, 7, 9 and 13 hours after initial treatment with 250 μM neomycin for 1 h. Panels A & B are posterior halves of zebrafish larvae (anterior to the left, dorsal up), where arrows indicate labeled neuromasts. Panels C, E, G and K are high magnification images viewed from probe 1 (λ_ex at 405 nm); panels D, F, H and L are from 4-Di-2-ASP (λ_ex at 488 nm).

Scheme 1.

Structures of commercial styryl dyes and compound 1, along with their absorption and fluorescence.

Scheme 2.

Schematic illustration of formation of the keto form via excited state intramolecular proton transfer (ESIPT). And the intramolecular charge transfer.