Berlin 2015 – scientific programme

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BP: Fachverband Biologische Physik

BP 7: Superresolution Optical Microscopy (focus session)

BP 7.6: Talk

Monday, March 16, 2015, 16:15–16:30, H 1028

Optical nanoscopy with self-healing fluorophores — Jasper H. M. van der Velde, Jingyi Huang, Andreas Herrmann, and •Thorben Cordes — Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

Customized buffer cocktails are so far the method of choice to facilitate photoswitching and to enhance signal stability for various implementations of optical super-resolution microscopy - a strategy that is not applicable for live-cell imaging.

In this contribution, we tested organic fluorophores with intramolecular photostabilization in STED-type microscopy to improve spatial and temporal resolution without using buffer additives. To obtain fluorophore-photostabilizer conjugates we use a recently published synthesis strategy based on unnatural amino acids. We explored the photostability and achievable spatial resolution of single ATTO647N-labelled oligonucleotides in STED imaging. We further tested KK114-conjugates for antibody labelling and STED imaging of the nuclear pore complex with the aim to increase the number of possible subsequent STED images. Finally, KK114-, Bodipy-Fl, and RhodamineB-derivatives were tested in STED-FCS. Our results show that intramolecular photostabilization is a simple and effective method to increase the photostability of dyes used for STED to achieve high spatial and temporal resolution. Strikingly, ATTO647N-photostabilizer conjugates allowed to generate single-molecule fluorescent time traces with excitation and STED-laser turned on, something that could so far not be achieved with standard fluorophores.