Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method

In two-color excitation fluorescence (2CE) theta microscopy, the point spread function (PSF) of the system is the product of the two PSFs of both excitation beams and due to the ellipsoidal shape of the PSFs, the axial width of the PSF of one excitation beam is strongly reduced after multiplication with the lateral section of another PSF in nonconfocal theta microscopy. Taking the advantages of this setup, we propose using two leaky filters inserted in the two illumination arms, respectively, to compress its own lateral width of each PSF; it means, the lateral and axial superresolution (three-dimensional superresolution) of the whole system in 2CE theta microscope with two orthogonal illumination beams are realized.