Determining the optimal expression method for dual-color imaging.

BACKGROUND: Fluorescence imaging is a widely used technique that permits for cell-type-specific recording from hundreds of neurons simultaneously. Often, to obtain cell-type-specific recordings from more than one cell type, researchers add an additional fluorescent protein to mark a second neuronal subpopulation. Currently, however, no consensus exists on the best expression method for multiple fluorescent proteins. NEW METHOD: We optimized the coexpression of two fluorescent proteins across multiple brain regions and mouse lines. RESULTS: The single-virus method, a viral injection in a double transgenic reporter mouse, results in limited fluorescent coexpression. In contrast the double-virus method, injecting a mixture of two viruses in a Cre driver mouse, results in up to 70 % coexpression of the fluorescent markers in vitro. Using the double-virus method allows for population activity recording and neuronal subpopulation determination. COMPARISON WITH EXISTING METHOD: The standard for expressing two fluorescent proteins is to use a double transgenic reporter mouse with a single viral injection. Injecting two viruses into a Cre driver mouse resulted in significantly higher coexpression compared to the standard method. This result generalized to multiple brain regions and mouse lines in vitro, as well as in vivo. CONCLUSION: Efficiently coexpressing multiple fluorescent proteins provides population activity while identifying a neuronal subpopulation of interest. The improved coexpression is applicable to a wide breadth of experiments, ranging from engram investigation to voltage imaging.