Diffusion-Matched and Spectrally-Discrete Lipophilic Probes for Neuronal Tracing∗

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Lipophilic fluorescent dyes enable the tracing of neural networks by diffusing laterally between nerve cell membranes. Because they do not require gene expression for labeling, these dyes can be advantageous for studies of both wild-type and mutant mice. To establish the neural connections that are made during development, a diffusion-matched set of spectrally distinct dyes is needed. A set with green, red and far-red fluorescence emission have previously been described [1]. Now, additional near-infrared and violet candidates have been developed. To optimize sequential six-color imaging protocols, we have measured the absolute multiphoton cross section spectra for these dyes. By combining two-photon and confocal microscopy, the entire set can be imaged using a single Ti:S laser. In the environment of a peripheral nerve fiber, the diffusion characteristics of dyes with varied hydrocarbon chain lengths and fluorescent head groups are determined by FRAP and distance measurements. By fitting the data to an anomalous-diffusion model, the time-scaling exponents and transport coefficients can be compared. Finally, we consider how the mechanism of lipophilic dye transport in fixed and living cells can be elucidated by transcellular diffusion from individually labeled cells within an interconnected network.[1] H. Jensen-Smith et al., IMMUNOL. INVEST, 36(5-6): 763-789, 2007.∗ This work was supported by an N.I.H. SBIR II grant, R44 MH079805-04, and by P20 RR016475 from the INBRE Program of the National Center for Research Resources.