Reflective Substrate-Enhanced Microsphere-Assisted Dark-Field Microscopy

Low-contrast small dielectric objects weakly interact with photons and are difficult to detect optically. In this study, we propose reflective substrate-enhanced microsphere-assisted dark-field microscopy which combines dark-field microscopy, silver-coated reflective substrate and microsphere to improve the resolution and contrast in imaging low-contrast objects. We demonstrate that the contrast in imaging low-contrast objects can be improved by replacing a microscope glass slide with a silver-coated reflective one. The angular distribution of the scattering signal of an object is modulated and the scattering signal that can be received by the objective is increased due to the strong coupling between the object and the reflective substrate. Moreover, the microsphere improves the resolution of the imaging system and the dark-field illumination provides high contrast images. As a result, the image intensity of a stand-alone polystyrene (PS) nanoparticle (150-300 nm in diameter) placed on a silver-coated slide is about 4.9 times of that of a same size PS nanoparticle placed on a glass slide under a conventional dark-field microscope without the microsphere. A 250-nm-diameter hexagonally close-packed PS nanoparticle monolayer can be discerned when the monolayer is placed on a sliver-coated slide under a microsphere-assisted dark-field microscope. However, when the same monolayer is placed on a plain glass slide and imaged by microsphere-assisted dark-field microscopy, or it is placed on a sliver-coated slide and imaged by dark-field microscopy, the monolayer cannot be observed. The proposed method is simple and easy to operate, which has potential applications in high resolution, high contrast, and low signal-to-noise ratio imaging of label-free biological samples.