Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams

Abstract

The confocal fluorescence microscope is the instrument of choice for biologists. However, compared to other instruments, its resolution is still limited. We propose a simple technique, based on laterally interfering beams, to improve the resolution. One technique consists in using a halve phase plate to modify the illumination, combined with a laterally offset detection. A 90nm lateral resolution is obtained for properly prepared specimens using readily available dyes. Another approach is to use several excitation beams, slightly shifted and properly dephased, to decrease the lateral extension of the PSF. With this approach, a lateral resolution of 75nm is predicted with the advantage of a regular confocal detection. Finally, we show how using these techniques in combination with a two-color two-photon excitation could permit to further improve the resolution to 60nm.