Single sepiolite clay needles doped with dyes of different molecular dimensions, Rhodamine 6G (R6G), Pyronine Y (PY), Styryl 698 (LDS 698) and Styryl 722 (LDS 722), at low and high loadings, are studied by confocal fluorescence microscopy. The combination of fluorescence lifetime imaging (FLIM), polarized fluorescence intensity experiments and spectral resolution shows a straightforward method to study the molecular distribution inside the tunnels and/or at the external channels, the dye orientation (evaluated by the fluorescence dichroic ratio, D) and the formation of different dye species (monomer and aggregates). The ability of the dyes to penetrate into the Sep tunnels is R6G < LDS 698 < PY < LDS 722, analyzed by thermal analysis. As a result the alignment along the main axis of Sep increases in the same order, reaching fluorescence dichroic ratios of around 1.5, 6, 8 and 10 respectively for each dye.