Long-term time-lapse fluorescence imaging of developing zebrafish

A variety of different transgenic zebrafish lines have been generated expressing green fluorescent protein (GFP) or other fluorescent proteins in different organs and tissues, permitting dynamic visualization of development of these organs and tissues in living animals via time-lapse imaging. Although methods have been devised for short-to medium-term time-lapse imaging of transgenic zebrafish, these methods are not suitable for longer term imaging because of poor control over temperature, evaporation, and anoxia. We describe a new imaging chamber that provides continuously circulating flow of warm, oxygenated aqueous media. We show that the chamber can be used for multiphoton time-lapse imaging of developing blood vessels in the trunk of Fli1-EGFP transgenic zebrafish for 5 days without developmental delay, loss of viability, or evident reduction in strength of circulatory flow. This imaging chamber provides an important new tool for long-term dynamic imaging of transgenic zebrafish.