Figure 1. NeuroPAL Method and Images.

(A) The emission for five distinguishable fluorophores. Each fluorophore’s excitation wavelength is listed in parentheses.

(B) Fluorophores are converted into pseudo colors to construct a primary color palette. Three fluorophores are designated as landmarks and pseudo colored to construct an RGB color palette: mNeptune2.5 is pseudo-colored red, CyOFP1 is pseudo-colored green, and mTagBFP2 is pseudo-colored blue. The fluorophore TagRFP-T is used as a panneuronal marker. The fluorophores GFP/CFP/YFP/GCaMP are reserved for reporters of gene expression or neuronal activity. TagRFP-T and GFP/CFP/YFP/GCaMP are visualized separately from the RGB landmarks to avoid confusion. They can be assigned any pseudo color.

(C) An example of how to stably pseudo color neurons, across animals. A set of reporters (rows), with stable neuronal expression (columns), are used to drive the fluorophores (table elements). NeuroPAL colors (last row) result from the combined patterns of reporter-fluorophore expression. The panneuronal reporter is expressed in all neurons. The remaining reporters have differential neuronal expression patterns and are used to drive the pseudo-colored landmark fluorophores. Combinations of these differentially expressed reporters assign stable and distinguishable pseudo colors to neurons. For example, reporter 3 drives the red landmark fluorophore (mNeptune2.5), to color neurons 2–4 with distinguishable red intensities and contributes to the blended coloring of several other neurons. In contrast, neuron 1 does not express any of the landmark fluorophores but is still marked by the panneuronal reporter.

(D) NeuroPAL scales this concept to 41 reporters that, in combination, disambiguate every neuron in C. elegans and thus generate, a single stereotyped color map across all animals (see Table S1 for data). Seven of the NeuroPAL reporters use a self-cleaving peptide sequence (T2A) to simultaneously drive expression of two different colors.

(E) Young adult NeuroPAL worms have a deterministic color map that remains identical across all animals (see Videos S1 and S2 for 3D images). Each neuron is distinguishable from its neighbors via color. All worm ganglia are shown.

All images may employ histogram adjustments to improve visibility. Images without a transmitted light channel (e.g., Nomarski) may further be adjusted with a gamma of ~0.5 to improve visibility on the dark background.

See also Table S1 and Videos S1 and S2.

See Figures S1 and Table S2 for phenotypic analysis.