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In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives

Kohei Watanabe16, Yuhei Nishimura1234, Takehiko Oka1, Tsuyoshi Nomoto6, Tetsuo Kon1, Taichi Shintou6, Minoru Hirano1, Yasuhito Shimada1234, Noriko Umemoto1, Junya Kuroyanagi1, Zhipeng Wang1, Zi Zhang1, Norihiro Nishimura25, Takeshi Miyazaki6, Takeshi Imamura6 and Toshio Tanaka1234*

Author affiliations

1 Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan

2 Mie University Medical Zebrafish Research Center, Tsu, Mie 514-8507, Japan

3 Department of Medical Chemogenomics, Mie University Venture Business Laboratory, Tsu, Mie 514-8507, Japan

4 Department of Bioinformatics, Mie University Life Science Research Center, Tsu, Mie 514-8507, Japan

5 Department of Translational Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan

6 Corporate R&D Headquarters, Canon Inc., Ohta-ku, Tokyo 146-8501, Japan

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Citation and License

BMC Neuroscience 2010, 11:116  doi:10.1186/1471-2202-11-116

Published: 15 September 2010



The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.


We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.


The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.