Photo- and Bio-physical Characterizations of Lipophilic Dyes for Neuronal Tracing
Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally between nerve cell membranes. Given the large number of connections that a typical neuron makes with its neighbors, a diffusion-matched set of spectrally distinct dyes is desirable. Previously, a trio of dyes was developed with well-separated green, red and far red fluorescence emission that permitted triple labeling. To extend this set to five, we have been characterizing the properties of novel blue and near-infrared candidates. By combining twophoton and confocal microscopy all of these candidates can be imaged using a single Ti:S laser. In order to develop a well-matched set of dyes, each dye must diffuse at the same rate across nerve cell membranes. Lipophilic dyes contain a head group and two hydrocarbon tails. By changing the head group, we get the various spectral properties needed to image multiple dyes at a time. The head group also affects the diffusive properties of the dye through the membrane. In order to compensate for these variations, we adjust the hydrocarbon tail length to diffusionmatch our dyes. Preliminary results on the rate of diffusion of these novel lipophilic dyes with varying hydrocarbon tails will be presented.