Fluorescence enhancement and reflection of the excitation light observed with a multilayered substrate

Fluorescence enhancement from a multilayered substrate fabricated with Ag and Al₂O₃ was investigated using fluorescein, rhodamine B, Cy3, and Cy5 as fluorophores. The change in the fluorescence enhancement with Al₂O₃ had two peaks and one valley in the range from 0 to 300nm of Al₂O₃ thickness, and such peaks and valley were found to appear periodically. Moreover, the reflection of the excitation light from the multilayered substrate was investigated. The reflection of the excitation light periodically changed depending on the Al₂O ₃ thickness as well, and the maximum reflection was observed near the Al₂O₃ thickness of the peak fluorescence enhancement. It was found that the periodic changes of the fluorescence enhancement and the reflection of the excitation light could be explained, for the most part, with the integral multiples of the λ/4 derived by a simple interference theory.