Three-color imaging using fluorescent proteins in living zebrafish embryos

The zebrafish embryo is especially valuable for cell biological studies bec ause of its optical clarity. In this system rise of an in vivo fluorescent reporter-has been limited to gl-een fluorescent protein (GFP). We have exam ined other fluorescent proteins alone or in conjunction with GFP to investi gate their efficacy as markers for multi-labeling purposes in live zebrafis h. By injecting plasmid DNA containing fluorescent protein expression casse ttes, we generated single; double-, or triple-labeled embryos using GFP, bl ue Florescent protein (BFP, a color-shifted GFP), and red fluorescent prote in (DsRed, a wild-type protein structure related to GFP). Fluorescent imagi ng demonstrates that GFP and DsRed are highly stable proteins, exhibiting n o detectable photoinstability, and a high signal-to-noise ratio. BFP demons trated detectable photoinstability and a lower signal-to-noise ratio than e ither GFP or DsRed. Using appropriate filter sets, these fluorescent protei ns can be independently detected even when simultaneously expressed in the same cells. Multiple labels in individual zebrafish cells open the door to a number of biological avenues of investigation, including multiple, indepe ndent tags of transgenic fish lines, lineage studies of wild-type proteins expressed using polycistronic messages, and the detection of protein-protei n interactions at the subcellular level using fluorescent protein fusions.