Intelligent-illumination STED.

Recently established fluorescence superresolution microscopy techniques, such as stimulated emission depletion (STED), are capable of imaging fixed and living cells at the nanometer scale. In STED, ON- and OFF-switching of fluorophores is performed with an excitation and a STED laser beam, respectively. During each switching cycle a finite probability exists that the fluorophore is photobleached. This hampers the possible STED resolution and sample structure definition. In this thesis, the connection between photobleaching, the obtainable fluorescence signal and the STED resolution was investigated. It was found that the maximum fluorescence signal extractable from a sample is inversely proportional to the STED resolution increase to the power of four. As a reverse conclusion, a higher fluorescence signal will always facilitate a better STED resolution and sample structure representation. To achieve such higher signals, a STED microscope was built which uses a spatial light modulator (SLM) as the phase mask element for the OFF-switching pattern creation. Especially under live-cell conditions, and at great sample depths, the SLM has conceptual advantages compared to conventional elements. To push the maximum fluorescence signal and resolution further, novel intelligentillumination scan schemata were developed. Those locally reduce photobleaching in the sample. The MINFIELD scan scheme (sub-diffraction sized scan fields of ~ 50−200 nm) was realized with 2D- and 3D-STED using intelligent beam control, leading to a simple microscope design with a great application range. The novel illumination scheme, Dynamic intensity MINimum (DyMIN), combines the advantages of MINFIELD and of the sample responsive illumination concept RESCue. With DyMIN, just as little OFF-switching intensity is applied to the current sample structure to have a clear ON/OFF-separation of the fluorophores. The typically much lower light dose provides a superior bleaching reduction for a large scan field. With 2D- and 3D-STED DyMIN scanning, a reduction of the light dose acting directly on the fluorophores up to 20-fold was possible, and the overall illumination of the sample was lowered more than 100-fold for sparser samples, both compared to a similar conventional scan. DyMIN enabled the best ever reported STED resolutions for a large field of view, ~ 17nm in the lateral direction, and ~ 34nm in the axial direction. I