Fluorescence emission difference with defocused surface plasmon-coupled emission microscopy.

ABSTRACT

A novel fluorescence emission difference method is proposed to improve the lateral resolution of SPCEM without increasing instrument complexity. We discovered the profile of transverse PSF in SPCEM will dramatically change from a hollow spot to a solid spot, when the axial position of sample varies within one wavelength in the vicinity of the focal plane. The subtraction of an image whose PSF is hollow spot and an image with solid PSF will greatly enhance the resolution and contrast of SPCEM images. The mechanism of the distinctive PSF is demonstrated through basic optics theories, and the improvement of lateral resolution is verified by theoretical simulations and experimental results. It is believed that our method will stand out for its pleasant resolution enhancement and its instruments' simplicity to facilitate many biological cellular observations.