Diffusion Measurements of Lipophilic Fluorescent Probes in Fixed Tissue and Living Cells

Abstract

By diffusing laterally along cell membranes, lipophilic fluorescent dyes delineate the neural pathways of both wild-type and mutant models. Multicolor imaging studies using a spectrally distinct set of diffusion-matched dyes are needed to further develop our understanding of complex neuronal connections. Previously, a set of dyes with fluorescence emission ranging from the UV to NIR was characterized and used to demonstrate six-color neuronal tracing. Using FRAP and relative distance measurements, transcellular diffusion in fixed tissue was shown to depend on the fluorescent head group. Now to compensate for this head-group-dependent diffusion, the influence of the hydrocarbon chain length has been characterized. Time-scaling exponents and diffusion coefficients within peripheral nerve tissue were compared to measurements in living cell culture. Surprisingly, it was found that the diffusion rates along the nerve increased with increasing hydrocarbon chain length. To elucidate the mechanism of lipid diffusion between cells, additional relative diffusion measurements in cultured living cells were performed by labeling a single cell within an interconnected network and measuring the spread of the fluorescent probe into surrounding cells. Taken together, these studies provide a systematic approach for the design of spectrally-discrete and diffusion-matched fluorescence probes for neurotracing.