Evanescent-Field Microscopy Combining Resonant Enhancement with Extended Penetration Depth

Abstract

Evanescent-field imaging enables selective probing of near-interface regions and has been widely used to visualize cell membrane dynamics, cell–substrate interfaces, nanoparticles, and surface-associated biological structures. Although conventional plasmonic illumination enables sensitive detection of subtle changes in the local dielectric environment through resonant field enhancement, its effective probing range is typically confined to within 100–200 nm of the interface. Here, we present long-range plasmonic illumination microscopy (LRPIM) based on a metal–dielectric multilayer chip. At the sample-facing surface, the chip generates an enhanced evanescent field that penetrates deeply into the sample while preserving low-background near-field excitation. Using individual microspheres as scattering probes, we established a calibration relationship between the evanescent-field penetration depth and the full width at half-maximum (FWHM) of the focused hotspot formed at the exit surface of each microsphere. Based on this calibration, the chip exhibited an electric-field penetration depth approximately 4.3 times that obtained with plasmonic scattering microscopy (PSM), enabling near-surface illumination over a micrometer-scale axial range. Furthermore, LRPIM supports fluorescence imaging, enhancing fluorescence signals from propidium iodide (PI)-stained cells and revealing localized nucleic acid enrichment within their nuclei. We anticipate that LRPIM will provide a straightforward chip-based approach to evanescent-field scattering and fluorescence microscopy, enabling structures to be probed over a micrometer-scale axial range near interfaces.