Two-photon fluorescence isotropic-single-objective microscopy

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.