This post was contributed by Richi Sakaguchi from Kyoto University, and Marcus N. Leiwe and Takeshi Imai from Kyushu University.
Stochastic multicolor labeling is a powerful solution for discriminating between neurons for light microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP system to express one of the three fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity will result in a combinatorial expression of these three genes with different copy numbers, producing dozens of color hues (Livet et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level.