STED Microscopy with Scanning Fields Below the Diffraction Limit

Since its development, STED microscopy has been used extensively for imaging biological samples. To label structures of interest, fluorescent proteins and organic dyes are used almost exclusively. However, these labels undergo irreversible photobleaching reactions when illuminated, which limits the available signal and ultimately the feasible resolution. This thesis presents a new approach to increase the signal yield in STED microscopy and related techniques. In these methods, the area that contributes to the fluorescence signal is much smaller than the illuminating laser foci. Thus, the irradiation dose per dye molecule can be significantly reduced by confining the image size to below the illuminated area. The probability of a photobleaching event during one image frame is reduced and more signal can be acquired. A STED microscope specialized for small scan areas was built. As only few sample points are imaged, the frame acquisition time is in the order of ten milliseconds. High scan frequencies would lead to oscillations in mechanical beam scanners. Therefore, it was necessary to use electro-optic beam deflectors to guarantee accurate positioning of the laser foci. A software was developed to select the desired scan-area positions from an overview image, which is acquired with a piezo-scanning stage. The viability of this approach was demonstrated on a variety of samples. It was shown on the dye Atto647N that the bleaching rate scales approximately linear with the STED intensity. Thus, the profit gained by reducing the scan area increases with the used STED laser power. In the limit of zero STED power, the photon yield of a fluorophore would be unchanged. Furthermore, the structural details in the focus area would not be resolvable. When increasing the resolution with STED to 20nm, however, the reduction of the scan area to 70x70nm2 yields 100 times more signal as compared to a regular scan size. This makes the developed method especially useful for investigating structures smaller than 200 nanometers. As an example, the nucleoporins NUP98 and NUP93 were imaged at a resolution below 20nm, revealing their arrangement in the nuclear pore