Deep-hole array structure for highly sensitive fluorescence imaging of cells via inverted microscopy

In this study, a plasmonic chip with a deep-hole array (DHA) structure enhanced fluorescence, enabling highly sensitive detection of targets via inverted fluorescence microscopy. Inverted microscopes are particularly suitable for detecting membrane proteins at the cell-substrate interface, where the cell membrane spreads in a single plane. Fluorescence enhancement should be homogeneous for high-sensitivity imaging across a wide field of view. However, in surface-plasmon-mediated enhancement, a periodic nanostructure covered with a thin metal film typically reduces the transmitted light and hinders observation via inverted microscopy. Therefore, we used a periodic DHA with a depth of 422 nm. The deep-hole structure enlarges the holes' inner areas, including the sidewalls, thus effectively reducing the metal thickness within the holes. The resulting increase in optical transmittance improved the fluorescence intensity detected by an inverted microscope. Although the transmittance of the DHA structure remained below 10% compared with that of a cover slip, it was more than 50-fold higher than that of the conventional hole-array structure (30 nm depth), resulting in an approximately 70-fold higher fluorescence intensity in observations of fluorescent particles with 0.2 µm diameters. The fluorescence enhancement achieved by the DHA plasmonic chip was further confirmed using biological samples. The imaging of breast cancer cells labeled with APC-EpCAM antibodies demonstrated that EpCAMs distributed on the cell membrane at the substrate interface, which were not discernible on a cover slip, were clearly detectable with high sensitivity. These findings establish the proposed DHA plasmonic chip as a powerful platform for highly sensitive fluorescence detection with potential applications in single-nanoparticle analysis and cellular bioimaging.