Photo- and bio-physical characterization of novel blue and near-infrared lipophilic fluorophores 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 [1]. To extend this set to five, we have been characterizing the properties of novel blue and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Ti:S laser. Here we present measurements of the absolute two-photon excitation spectra along with single- and two-photon fluorescence recovery after photobleaching measurements of the diffusion coefficient in spinal cord samples. [1] H. Jensen-Smith et al., Immunol. Invest., in press, 2007.