Spectrum change and intensity fluctuation of the fluorescence emitted by single pyridine2 (1-ethyl-4-(p-dimethylaminophenyl)1,3-butadienyl)-pyridinium perchlorate; Py2) molecules adsorbed on a glass plate were measured at room temperature in real time by simple modi. cation of a conventional optical microscope, i.e., by incorporating a transmission grating. The spectral shapes and peak wavelengths have wide distribution. The spectra could be classified into three distinct types. Most of the molecules exhibited one of the three types until they were finally photobleached, while some molecules showed occasional spectral jump to another type, which was found to be reversible. These results can be ascribed to the presence of several different conformers and photochemical isomerization. (c) 2008 Elsevier B.V. All rights reserved.