Enhancing the axial resolution of fluorescence microscopy using two-photon confocal configuration

Abstract

We increase the axial resolution of a fluorescence microscope by utilizing two-photon excitation in a confocal configuration. This resolution increase is rooted on the nonlinear nature of the excitation process which was verified to have a power dependence of 2.02 – the exact excitation signature of a two-photon absorption process and on the detection discrimination in provided by the confocal pinhole. The two-photon confocal arrangement improves the resolution to 1.6 μm, 3 times sharper than two-photon widefield configuration and 96 times sharper than the single photon widefield case. The microscope is used to image 10 μm fluorescent spheres with minimal photobleaching.