High-contrast fluorescence imaging based on the polarization dependence of the fluorescence enhancement using an optical interference mirror slide.

High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

Highly sensitive fluorescence detection of avidin/streptavidin with an optical interference mirror slide.

This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

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

Fluorescence enhancement from a multilayered substrate fabricated with Ag and Al(2)O(3) was investigated using fluorescein, rhodamine B, Cy3, and Cy5 as fluorophores. The change in the fluorescence enhancement with Al(2)O(3) had two peaks and one valley in the range from 0 to 300 nm of Al(2)O(3) 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 Al(2)O(3) thickness as well, and the maximum reflection was observed near the Al(2)O(3) 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 lambda/4 derived by a simple interference theory.

Effect of the polarization and incident angle of excitation light on the fluorescence enhancement observed with a multilayered substrate fabricated by Ag and Al2O3.

The fluorescence from a fluorophore on a multilayered substrate fabricated by a metal and a dielectric is known to be enhanced by more than 100-fold. In the course of this study, we prepared a multilayered substrate with Ag as the metal and Al(2)O(3) as the dielectric and then investigated the effects of the polarization of the excitation light on the enhancement of the multilayered substrate. It was found that the enhancement was attributed to an electric field oscillating parallel to the substrate. Maximum 200-fold enhancement could be achieved with 80 nm thick Al(2)O(3) when an unpolarized excitation light was used with an incident angle of 20 degrees.