Abstract
Lipophilic fluorescent dyes are used to trace neuronal connections because of their ability to insert into and di use laterally within cellular membranes. These dyes successfully define neuronal tracts for normal and mutant development no matter how aberrant the molecular processing of cells. Simultaneous delineation of multiple pathways requires multicolor labeling with a set of diffusion matched and spectrally resolvable dyes. To achieve more than 3 distinct colors within a single sample, imaging strategies using two-photon excitation are necessary. The efficient utilization of two-photon excitation is dependent on the knowledge of the two-photon excitation action cross section for the set of dyes. The first specific aim of this thesis was to measure the two-photon excitation action cross-section spectra for a set of neurotracing dyes to facilitate the design of multicolor imaging strategies. Two-photon excitation action cross sections were measured using a standard ratiometric approach comparing the neurotracer dye fluorescence to a well-known dye. The results indicated that all fluorescent dyes measured can be efficiently two-photon excited with a Ti:S laser. The acquisition of the two-photon excitation spectra for the neurotracer set allowed dyes exhibiting fluorescence emission cross talk to be spectrally resolved, and thus achieve multicolor neurotracing.|Recently certain lipids have been implicated as signaling molecules that travel between cells. Although their effects are fairly well-characterized, the mechanism for their diffusion across the aqueous gap between cells is unknown. This mechanism might be affected by variations in the length of their hydrocarbon chains. Because lipophilic fluorescent dyes consist of a head group and two variable-length hydrocarbon chains analogous to naturally occurring lipids, they can be used to investigate lipid diffusion between living cells. The second specific aim of this thesis was to investigate the hydrocarbon-chain-length dependence on the mechanism of lipid transcellular di usion. Dye diffusion was examined by labeling single cells in a connected network and measuring the spread of dye into the network using laser scanning microscopy. Additionally, FRAP measurements within individual cells were performed. The results demonstrated that for both transcellular and intracellular diffusion, the length of the hydrocarbon chain influences the mechanism of lipid diffusion in living cells.