Dr. Matthias Reuss, Cell Physics, SciLifeLab, KTH

Abstract

Fluorescence microscopy is one of the most extensively used tools for the structural and functional investigation of the interior of cells despite the fact that it is limited by diffraction and thus fails to image structures smaller than ~200 nm. In 1994, however, with the invention of Stimulated Emission Depletion Microscopy (STED), the diffraction barrier was effectively broken. Today, STED microscopy provides nanometer scale resolution, while retaining most of the advantages of far-field optical operation, such as the ability to non-invasively image live cells.

 STED microscopes usually employ a scanned excitation beam superimposed by a donut-shaped STED beam that keeps fluorophores in their ground state except in its dark center. This way, the size of the effective excitation spot is dramatically reduced and the resolution increases accordingly. Nevertheless, practical difficulties that often arise stem from the fact that two separate beams have to be precisely overlaid and kept stable.

 Here, I will present easySTED, a method that provides intrinsic alignment. To this end, all beams are pre-combined in an optical fiber and then fed through a beam shaper that only affects the STED light and turns it into a donut while it keeps the excitation beam intact. An easySTED-system cannot physically go out of alignment and thus does not require optics experience for its operation.

 I will give a short introduction into super-resolution microscopy, cover the principle of easySTED with examples and highlight planned developments such as three-color and three-dimensional super-resolution imaging.