High-contrast fluorescence microscopy for a biomolecular analysis based on polarization techniques using an optical interference mirror slide

Fluorescence microscopy with an improved contrast for fluorescence images is developed using an optical interference mirror (OIM) slide, which can enhance the fluorescence from a fluorophore as a result of the double interference of the excitation light and emission light. To improve the contrast of a fluorescence image using an OIM slide, a linearly-polarized excitation light was employed, and the fluorescence emission polarized perpendicular to the polarization of the excitation light was detected. The image contrast with this optical system was improved 110-fold for rhodamine B spotted on the OIM, in comparison with a glass slide using a general fluorescence microscopy optical system. Moreover, a 24-fold improvement of the image contrast was achieved for the detection of Cy3-labeled streptavidin bound to immobilize biotin.