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.
Keywords: biochip; fluorescence enhancement; fluorescence microscopy.