In this study we attempt to determine the deactivation mechanism of a hybridization-sensitive DNA probe by using steady state absorption and fluorescence spectroscopy. Thiazole orange or 2,2'-Methylenebis(N-alkylbenzothiazole) is covalently linked to a single nucleotide in the DNA probe. Radiationless deactivation of the fluorophores is mainly determined by photo-induced isomerization of the methine bridge. Experiments done using solutes of differing molecular weight (sucrose, glucose and glycerin) to provide solutions of similar viscosity reveals the importance of solution viscosity and fluorophore conformation on radiationless deactivation. The quantum yield of the fluorophore in high viscous solutions can be explained by a simple diffusion model. This linear relationship is broken when a dimer conformation of the fluorophore starts to form, which in turn influences the emission intensity of the fluorophore. (C) 2012 Elsevier B.V. All rights reserved.