Bright multicolor labeling of neuronal circuits with fluorescent proteins and chemical tags

Elife. 2018 Nov 20;7:e40350. doi: 10.7554/eLife.40350.


The stochastic multicolor labeling method 'Brainbow' is a powerful strategy to label multiple neurons differentially with fluorescent proteins; however, the fluorescence levels provided by the original attempts to use this strategy were inadequate. In the present study, we developed a stochastic multicolor labeling method with enhanced expression levels that uses a tetracycline-operator system (Tetbow). We optimized Tetbow for either plasmid or virus vector-mediated multicolor labeling. When combined with tissue clearing, Tetbow was powerful enough to visualize the three-dimensional architecture of individual neurons. Using Tetbow, we were able to visualize the axonal projection patterns of individual mitral/tufted cells along several millimeters in the mouse olfactory system. We also developed a Tetbow system with chemical tags, in which genetically encoded chemical tags were labeled with synthetic fluorophores. This was useful in expanding the repertoire of the fluorescence labels and the applications of the Tetbow system. Together, these new tools facilitate light-microscopy-based neuronal tracing at both a large scale and a high resolution.

Keywords: Brainbow; chemical tag; fluorescence imaging; mouse; neuronal circuit; neuronal tracing; neuroscience; tissue clearing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Neuroanatomical Tract-Tracing Techniques / methods*
  • Neuronal Tract-Tracers / metabolism*
  • Olfactory Bulb / anatomy & histology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staining and Labeling / methods*


  • Luminescent Proteins
  • Neuronal Tract-Tracers
  • Recombinant Proteins

Grants and funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.