Saturated two-photon excitation fluorescence microscopy with core-ring illumination.

Abstract

We demonstrated resolution improvement in two-photon excitation microscopy by combining saturated excitation (SAX) of fluorescence and pupil manipulation. We theoretically estimated the resolution improvement and the sidelobe effect in the point spread function with various pupil designs and found that the combination of SAX and core-ring illumination can effectively enhance the spatial resolution in 3D and suppress sidelobe artifacts. The experimental demonstration shows that the proposed technique is effective for observation with a depth of 100μm in a tissue phantom and can be applied to 3D observations of tissue samples with higher spatial resolution than conventional two-photon excitation microscopy.