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To establish a calibration method of optical performance in fluorescence microscopy, we fabricated a fluorescent nanometer-scale marker by combining a dry dye method for polymer film and fine lithography. The marker has a 50 nm line-and-space fluorescent pattern, finer than the optical diffraction limit. A spin-coated poly͑methyl methacrylate͒ thin film on a silicon wafer was densely doped with Rhodamine 6G using a simple vacuum process, named the vapor-transportation method, and then the pattern was formed on the film using electron-beam lithography. The figure accuracy of the fabricated marker was calibrated by electron microscopes. Using this marker, one can quantitatively evaluate the optical properties; i.e., the contrast-transfer function, the point-spread function, magnification, and so on. To show practical use of the marker, we demonstrated the evaluation of a fluorescent microscope system.

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