Breaking the diffraction limit of light sheets allows fast isotropic imaging of large samples by ultramicroscopy

Abstract

By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to generate extremely long thin sheets of light with a thickness in the one micron range and a vastly increased Rayleigh range. We measured the thickness of the light sheets with different methods including standard point spread function measurement with beads. By using these light sheets in our ultramicroscope fast 3D imaging of whole mouse brains with objectives with a large field of view was possible. Due to the extremely low divergence of the light sheets mouse brains could be reconstructed from a single stack of optical sections with isotropic resolution. The light sheets used were essentially non-Gaussian generated by new optics we developed. Compared to a Gaussian light sheet of the same NA our new light sheet is much thinner. Thus the diffraction limit which holds also for low NA Gaussian light sheets was significantly surpassed. These optics will allow the application of ultramicroscopy to ever increasing samples beyond the whole mouse brain range.