A Novel STED Microscope with Nanometer Axial Sectioning

Abstract

The axial resolution of conventional Stimulated emission depletion (STED) microscopy is limited by diffraction to about 500 nm. Overcoming this limitation usually comes at the cost of complex instrumentation, as 3D confinement usually implies to combine two STED beams. By trying to combine with TIRF excitation this decrease the lateral performances. In this work we reported a straightforward approach to restrict the axial extension of the STED microscope by only modifying the detection path, thus keeping optimal lateral resolution. This new membrane imaging takes advantage of the supercritical angle (SAF) emission of fluorophore that allows one to discriminate their position regarding the glass coverslip. Indeed only when fluorophore are closed to the interface, their evanescent near field can become propagative and appears above the critical angle. This SAF emission represents up to 50% of the emission collected by the objective for a fluorophore at the interface. By filtering out of the undercritical emission in a conjugated plane of the back focal plane of the objective lens, only the SAF emission is detected. It allows nanometric axial sectioning of fluorescent emitters close to the water-glass interface; simply by adding a SAF module in the detection path of all STED microscopes. This STED-SAF implementation reduces the complexity and cost of its early implementation and as side effect, the photobleaching and light dose sent to the sample is reduced since not extra STED beam is needed for isotropic resolution. In this work we show for the first time the coupling of STED and SAF microscopy and we highlight the benefits of this implementation by imaging fluorescent beads and sub-cellular structures. The image quality of raw data is improved by using deconvolution techniques. Finally the specific importance of the method towards the implementation of Tomography-STED microscopy is discussed.