Zebrafish are a widely used developmental model because of their transparent embryos and external development. These distinctive characteristics provide valuable insights into embryonic development. Optical coherence tomography (OCT) offers label-free structural imaging and has emerged as a preferred tool for embryonic imaging. On the other hand, light sheet fluorescence microscopy (LSFM) enables time-lapse molecular imaging of multi-hour to multi-day developmental processes due to its low phototoxicity and photobleaching compared to traditional confocal fluorescence microscopy. We developed a multimodal imaging system to obtain concurrent structural and molecular information by combining OCT and LSFM for embryonic imaging. A Michelson-type swept-source OCT system with a central wavelength of 1050 nm, the bandwidth of 100 nm, and sweep rate of 100 kHz captured the structural information with a lateral resolution of ~15 μm and an axial resolution of ~7 μm. The LSFM system captured the molecular information with a transverse resolution of ~2.1 μm and an axial resolution of ~13 μm. The optically co-aligned OCT and LSFM beams were scanned through the same scan head for trivial co-registration of the multimodal images. We imaged 1-5 μm green fluorescence microbeads to show the capability of this system. We then conducted imaging of zebrafish vasculature development with a transgenic line, Tg(kdrl:EGFP), where the erythroblasts express GFP. The results show that the multimodal system enables us to provide co-registered zebrafish structural and functional imaging.
Phenylthiourea (PTU) is often used to block pigmentation and make zebrafish completely transparent for easy optical imaging. PTU inhibits melanogenesis by inhibiting tyrosinase. Although the PTU is commonly used, it does have some side effects. PTU at a concentration of 0.2 mM (0.003%) significantly reduces the zebrafish eye size due to the inhibition of thyroid hormone production. Furthermore, low levels of thyroid hormones in zebrafish increase the stiffness of the intervertebral joints, altering their swimming behavior. The aim of this study was to assess the structural modifications and biomechanical properties of 5-day post-fertilization (dpf) zebrafish eyes after being exposed to PTU using optical coherence tomography and reverberant optical coherence elastography, respectively. Wild-type zebrafish (n=3), treated with PTU (0.2 mM), were compared with non-treated zebrafish (n=3). The results show a significant reduction (p=0.02) in the mean eye diameter of the fishes treated with PTU (312.66 ± 8.71 μm) versus the non-treated group (340.18 ± 4.38 μm). On the other hand, the non-treated group showed a significantly slower (p=0.02) shear wave speed (0.97 ± 0.12 m/s) compared with the PTU-treated group (2.65 ± 0.51 m/s), indicating that PTU induces a biomechanical change in the stiffness of the developing eye. PTU is a potent inhibitor of the pigmentation of zebrafish; however, it can also severely affect its biomechanical properties, specifically eye development, reducing eye diameter and increasing its stiffness.
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