Fabrication of nanostep for total internal reflection fluorescence microscopy to calibrate in water

• Nanostep, whose refractive index was close to that of water, was obtained.
• The nanostep was fabricated via lift-off technique using PVA film and metal mask.
• The metal mask was transferred from a resin replica mold via nano transfer printing.
• The nanostep was made of a polymer resin and it was invisible in water.
• Using the nanostep, we succeeded in the calibration of the TIRF intensity in water.

Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for analyzing x–y–z (3D) fluid flow in nano- or micro-channel. To visualize the 3D fluid flow via TIRFM, intensity calibration of a fluorescent particle is required by using calibration plates, carrying a set of nano-steps of different heights. In an attempt to fabricate a calibration plate to be used in water ambient 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 resin pattern on a glass substrate by employing the use of polyvinyl alcohol film and metal mask previously fabricated by nano transfer printing (nTP). In summary, we have succeeded in the fabrication of nanosteps made of a polymer resin and used it for the calibration of the fluorescence intensity in water.

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