Abstract
Total internal reflection fluorescence microscopy (TIRFM) has gotten a lot of attention because it is very useful for analyzing fluid flow in nano- or micro-channel. To calibrate the intensity of a fluorescent particle in water ambient, a nano-step pattern, whose refractive index is same to the working fluid, is needed. Therefore, we employed a polymer with a refractive index close to that of water. In this study, we have developed a lift-off technique for making polymer nano-step pattern on a glass substrate by using polyvinyl alcohol film and metal mask obtained by nano transfer printing (nTP). In summary, we have succeeded in the calibration of the fluorescence intensity of a particle in water.
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