Figure 1. Overview of chemical structures and properties of DiI and CLARITY–compatible analogues.

DiIC₁₈(3) (DiI in short) is a fluorescent lipophilic cationic indocarbocyanine dye used for single molecule imaging, fate mapping, and neuronal tracing, as it is retained in the lipid bilayers. CM-DiIC₁₈(3) (CM-DiI in short) incorporates a mildly thiol–reactive chloromethylbenzamido (CM) substituent (green) that confers aldehyde fixability via conjugation to thiol-containing proteins. SP-DiIC₁₈(3) (SP-DiI in short) has two sulfophenyl (SP) groups (magenta) that confers fixability and a greater ε than DiIC₁₈(3). FM 1–43FX, which is less similar to DiIC₁₈(3), is a lipophilic styryl dye with an aliphatic amine side chain (red) for aldehyde fixability. It has, notably the lowest ε, and the greatest Stokes shift, but also half the molar mass due to lacking the two 18-carbon-long alkyl tails (blue). *Spectral properties determined in methanol. ε: molar attenuation coefficient in cm⁻¹ M⁻¹.