04.12.2013 

Prof. Gerd Ulrich Nienhaus 

16:30 

A number of sophisticated fluorescence-based optical microscopy concepts have been introduced in recent years featuring image resolutions well below the diffraction (Abbe) limit. Consequently, they are appropriately referred to as optical nanoscopy. Mainly driven by the molecular imaging needs of the life sciences, efforts are ongoing to further advance these methods, importantly, to improve data acquisition speed and the extension to all three spatial dimensions. In this lecture, I shall present an overview of current techniques and include recent work from our laboratory.
Localization microscopy (PALM/STORM) is a widefield optical nanoscopy technique that is conceptually simple and easy to implement. By using photoactivatable fluorescent proteins, the technique allows the dynamics of live cells to be imaged [1]. Individual fluorescence emitters are activated stochastically by light and are subsequently localized with high precision (1 – 30 nm). The image quality hinges on the performance of the image reconstruction software [2]. By using GPU-based computation, fairly complex analysis algorithms can be implemented on PCs that still keep up with the data flow from state-of-the-art CCD or sCMOS cameras [3]. Data collection for a super-resolution localization image takes on the order of seconds; faster (millisecond) dynamics can be extracted from the data by analyzing single particle tracks.
STED/RESOLFT microscopy is a scanning technique that uses especially shaped focal patterns to sharpen the point-spread function, either by using stimulated emission of fluorophores or by reversible switching of photoactivatable fluorophores for spatially selective depletion of the excitation. Image acquisition scales with the number of pixels, so small subregions of interest can be imaged quickly. Fast dynamics such as molecular diffusion can be accessed by fluorescence fluctuation techniques such as fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS). For both methods, the combination with STED offers key advantages, particularly the extension of the marker concentration up to the micromolar range and the enhanced spatial resolution [4].

References
1. J. Fuchs, S. Böhme, F. Oswald, P. N. Hedde, M. Krause, J. Wiedenmann, and G. U. Nienhaus, Nat. Methods 7, 627-630 (2010).
2. P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, Nat. Methods 6, 689-690 (2009).
3. Y. Li, P. N. Hedde, Y. Ishitsuka and G. U. Nienhaus, ACS Nano 7, 5207-5214 (2013).
4. P. N. Hedde, R. M. Dörlich, R. Blomley, D. Gradl, E. Oppong, A. C. B. Cato and G. U. Nienhaus, Nat. Commun. 4, 2093 (2013).