Imaging Neural Architecture in Brainbow Samples

Methods Mol Biol. 2017;1642:211-228. doi: 10.1007/978-1-4939-7169-5_14.


The fluorescent protein revolution has made the light microscope the most widely used tool for studying biological structure from the single-molecule to whole organism scales. However, traditional approaches are limited in their ability to resolve components in highly complex structures, such as the brain. In recent years, this limitation has been circumvented by the development of multicolor labeling methods, termed Brainbow. Brainbow tools rely on site-specific recombinases to make stochastic "choices" between different combinations of fluorescent proteins so that structures in close proximity to one another can be resolved based on their color profile. These new approaches, however, call for more refined methods of sample preparation and imaging optimized for multispectral imaging, which are presented here. The most robust approach for generating useful Brainbow data combines immunohistology with multispectral laser scanning confocal microscopy. This chapter, therefore, focuses on this particular technique, though the imaging principle discussed here is applicable to other Brainbow approaches as well.

Keywords: Brainbow; Confocal microscopy; Development; Immunohistology; Linear unmixing; Multispectral imaging; Neuroscience.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Brain / metabolism
  • Brain / ultrastructure*
  • DNA Nucleotidyltransferases / genetics
  • DNA Nucleotidyltransferases / metabolism
  • Dependovirus / genetics
  • Dependovirus / metabolism
  • Fluorescent Dyes / chemistry
  • Genes, Reporter
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Integrases / genetics
  • Integrases / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Microscopy, Confocal / methods
  • Nerve Net / metabolism
  • Nerve Net / ultrastructure*
  • Optical Imaging / instrumentation
  • Optical Imaging / methods*
  • Promoter Regions, Genetic
  • Recombination, Genetic*
  • Staining and Labeling / methods*
  • Zebrafish


  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Cre recombinase
  • DNA Nucleotidyltransferases
  • FLP recombinase
  • Integrases